Plants with altered color and methods of making same

Abstract

Provided is a plant in which the flower color of the plant is changed. The present invention relates to a method for producing a plant in which the flower color of the plant is changed, the method comprising accumulating an anthyanidin pigment in cells of petals of the plant in an amount of 30 mass% or more with respect to the total amount of pigment present in the cells, and accumulating the anthyanidin pigment in the cells in an amount of 30 mass% or more with respect to the total amount of pigment present in the cells in comparison to the flower color of the plant prior to accumulation of the anthyanidin pigment. The flower color of a plant after accumulation of an anthyanidin pigment is changed so that the value in the CIE color system is reduced.

Description

[Technical Field]

[0001] This invention relates to plants with altered flower color and methods for producing them. [Background Technology]

[0002] The floriculture industry is dedicated to developing novel and diverse varieties. One effective method for breeding new varieties is changing flower color. Using classic breeding methods, this has resulted in varieties with a wide range of flower colors for most commercial varieties.

[0003] To date, for example, Patent Document 1 has reported a method for producing plants with blue-green flowers. More specifically, the method of Patent Document 1 is a method for producing plants with blue-green flowers, characterized in that delphinidin-type anthocyanins with glycosylated positions 3' and 5' of the anthocyanin B ring coexist with flavonoid glycosides or flavonol glycosides within the plant cells.

[0004] In addition, Patent Document 2 discloses a novel Mandevilla with a new hue that has not been made until now. Mandevilla Plants of the genus *Mandevilla*. More specifically, the novel *Mandevilla* of Patent Document 2 (*Mandevilla spp.*) Mandevilla The genus *Mandevilla* refers to plants whose petals contain at least one type of carotenoid pigment. Mandevilla The plant is characterized by the fact that the carotenoid pigment is neolutein or its derivative.

[0005] [Existing Technical Documents]

[0006] [Patent Documents]

[0007] Patent Document 1: International Publication No. 2017 / 169699

[0008] Patent Document 2: Japanese Patent Application Publication No. 2021-175402

[0009] [Non-patent literature]

[0010] Non-patent literature 1: Tatsuzawa, F. et al. Biochem. Systemat. Ecol. 99, 2021, 104347

[0011] Non-patent literature 2: Macz-Pop, GA et al. Food Chem. 94, 2006, 448-456

[0012] Non-patent literature 3: Oyama, K. et al. J. Agric. Food Chem. 63, 2015, 7630-7635 [Summary of the Invention]

[0013] [The problem the invention aims to solve]

[0014] Development related to plants with changing flower colors is still needed.

[0015] For example, in Mandevilla ( Mandevilla Among plants of the genus *Mandevilla*, only varieties with white, pink, and red flowers have been found so far, although Patent Document 2 provides new colors of *Mandevilla* containing carotenoid pigments. Mandevilla Plants of the genus *Mandevilla*, but still require information on the green hues of the *Mandevilla* vine. Mandevilla Research on plants of the genus ().

[0016] The present invention aims to improve the above situation and its purpose is to provide a plant with altered flower color and a method for doing so.

[0017] [Solutions for solving the problem]

[0018] The present invention for achieving the above objectives is as follows.

[0019] [Implementation Method 1]

[0020] A method for producing plants, wherein the method includes:

[0021] In the cells of the petals of the plant, anthocyanin pigments accumulate at a rate of 30% or more by mass relative to the total amount of pigments present in the cells, and

[0022] Compared to the flower color of the plant before the accumulation of the anthocyanin pigment, the flower color of the plant after the accumulation of the anthocyanin pigment is as follows (CIE) Color system The way the value decreases has changed.

[0023] [Implementation Method 2]

[0024] The method described in Embodiment 1, wherein at least a portion of the anthocyanin-modifying enzyme gene is deleted, thereby causing the anthocyanin pigment to accumulate.

[0025] [Implementation Method 3]

[0026] The method described in Implementation Method 2, wherein,

[0027] The method includes deleting at least a portion of the promoter region of the anthocyanin-modifying enzyme gene, and

[0028] The anthocyanidin modifying enzyme is selected from: anthocyanidin glycosylation enzyme gene and / or anthocyanidin 3-glucosidase.

[0029] [Implementation Method 4]

[0030] The method described in Implementation Method 3, wherein,

[0031] The anthocyanin glycosylation enzyme is a galactosyltransferase, and

[0032] In the promoter region of the galactosyltransferase gene, at least one sequence selected from the following is deleted: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them.

[0033] [Implementation Method 5]

[0034] The method described in Implementation 3, wherein

[0035] The anthocyanidin 3-glucosidase is a xylose transferase, and

[0036] Insert at least one sequence selected from the following into the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity with it.

[0037] [Implementation Method 6]

[0038] The method of any one of embodiments 1 to 5, wherein the anthocyanidin pigment is cyanidin.

[0039] [Implementation Method 7]

[0040] The method of any one of embodiments 1 to 6, wherein the anthocyanin pigment is locally present in the cytoplasm of the cell.

[0041] [Implementation Method 8]

[0042] The method of any one of embodiments 1 to 7, wherein the cells of the petals of the plant contain chlorogenic acid and aluminum ions.

[0043] [Implementation Method 9]

[0044] The method of any one of embodiments 1 to 8, wherein the plant is selected from at least one of the following: Apocynaceae, Solanaceae, Solanaceae, and Scrophulariaceae.

[0045] [Implementation Method 10]

[0046] The method of any one of embodiments 1 to 9, wherein the plant is at least one selected from the following: Mandevilla ( Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia Plants of the genus ().

[0047] [Implementation Method 11]

[0048] The method of Embodiment 10, wherein the plant is a plant or its offspring deposited with accession number FERM ABP-22483 at the Patent Biological Collection Center (NITE-IPOD), an independent administrative agency for product evaluation technology.

[0049] [Implementation Method 12]

[0050] Plants, specifically, in the cells of plant petals,

[0051] Anthocyanin pigments accumulate to more than 30% by mass relative to the total amount of pigments present in the cells, and

[0052] At least a portion of the anthocyanidin-modifying enzyme gene is missing.

[0053] [Implementation Method 13]

[0054] The plant of embodiment 12, wherein the anthocyanin pigment is locally present in the cytoplasm of the cells.

[0055] [Implementation Method 14]

[0056] The plant described in embodiment 12 or 13, wherein,

[0057] The plant includes a portion of the promoter region of the anthocyanin-modifying enzyme gene that is deleted, and,

[0058] The anthocyanidin modifying enzyme is selected from anthocyanidin glycosylation enzyme and / or anthocyanidin 3-glucosidase.

[0059] [Implementation Method 15]

[0060] The plant described in embodiment 14, wherein,

[0061] The anthocyanin glycosylation enzyme is a galactosyltransferase, and,

[0062] In the promoter region of the galactosyltransferase gene, at least one sequence selected from the following is deleted: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them.

[0063] [Implementation Method 16]

[0064] The plant described in embodiment 14, wherein,

[0065] The anthocyanidin 3-glucosidase is a xylose transferase, and,

[0066] Insert at least one sequence selected from the following into the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity with it.

[0067] [Implementation Method 17]

[0068] The plant described in any one of embodiments 12 to 16, wherein the anthocyanidin pigment is cyanidin.

[0069] [Implementation Method 18]

[0070] The plant according to any one of embodiments 12 to 17, wherein the cells of the petals of the plant contain chlorogenic acid and aluminum ions.

[0071] [Implementation Method 19]

[0072] The plant described in any one of embodiments 12 to 18, wherein the plant is selected from at least one of the following: Apocynaceae, Solanaceae, Solanaceae, and Scrophulariaceae.

[0073] [Implementation Method 20]

[0074] The plant described in any one of embodiments 12 to 19, wherein the plant is selected from at least one of the following: Mandevilla (… Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia Plants of the genus ().

[0075] [Implementation Method 21]

[0076] The plant described in Embodiment 20, wherein the fragrant vine ( Mandevilla Plants of the genus FERM ABP-22483 are plants or their descendants deposited at the Patent Biological Collection Center (NITE-IPOD) with accession number FERM ABP-22483.

[0077] [Implementation Method 22]

[0078] The offspring of the plant described in any one of embodiments 12 to 21.

[0079] [Implementation Method 23]

[0080] The propagation material of the plant described in any one of Embodiments 12 to 21.

[0081] [Implementation Method 24]

[0082] A part of the plant body, tissue or cell of any one of embodiments 12 to 21.

[0083] [Invention Effects]

[0084] According to the present invention, a plant that alters the flower color can be provided. [Attached Image Description]

[0085] Figure 1 This is a graph showing the pigment analysis results in Example 1.

[0086] Figure 2 This is a graph representing the HPLC analysis results from Example 1.

[0087] Figure 3 This is a diagram showing the structural analysis results in Example 2.

[0088] Figure 4 This is a graph showing the expression analysis of the galactosyltransferase gene and xylose transferase gene in Example 3.

[0089] Figure 5 This is a graph showing the LC-MS analysis results in Example 4.

[0090] Figure 6 This is a graph showing the results of extraction using various solvents in Example 5.

[0091] Figure 7 This is a diagram illustrating the experiment of Example 6.

[0092] Figure 8 This is a diagram showing the analysis and alignment results of the base sequence of the galactosyltransferase gene in Example 7.

[0093] Figure 9 This is a diagram showing the analysis and alignment results of the base sequence of the xylose transferase gene in Example 7.

[0094] Figure 10 This is a diagram illustrating the design for creating gene markers in Example 7.

[0095] Figure 11 This is a graph showing the PCR amplification results in Example 7.

[0096] Figure 12 This is a diagram showing the location where the pigment in Example 8 is locally present within the cell.

Detailed Implementation Methods

[0097] The embodiments of the present invention are described in detail below. Furthermore, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the inventive spirit.

[0098] Methods for growing plants

[0099] The method for producing the plant according to the present invention (hereinafter sometimes simply referred to as "the method of the present invention") is as follows:

[0100] In the cells of plant petals, anthocyanin pigments accumulate at a rate of 30% or more by mass relative to the total amount of pigments present in the cells, and...

[0101] Compared to the flower color of plants before anthocyanin pigment accumulation, the flower color of plants after anthocyanin pigment accumulation is indicated by CIE [data missing]. Color system The way the value decreases changes.

[0102] To date, as described in Patent Document 1 above, there are technical concepts for accumulating anthocyanin pigments in glycoside form within petal cells. However, since pigments in their aglycone state are generally unstable and stabilized within cells through modifications such as glycosylation and acylation, no precedent has been reported for accumulating anthocyanin pigments, i.e., pigments in their aglycone state, within petal cells, or for accumulating anthocyanin pigments within the cells of plant petals.

[0103] Therefore, the present invention’s concept of accumulating anthocyanin pigment at a rate of 30% or more by mass relative to the total amount of pigment present in the cells of plant petals is novel. Furthermore, the results of Examples 1 to 8 described later indicate that the method of the present invention can accumulate anthocyanin pigment in the cells of plant petals (especially in the cytoplasm).

[0104] The method of the present invention enables the accumulation of anthocyanin pigment in the cells of plant petals at a rate of 30% by mass or more relative to the total amount of pigment present in the cells. More specifically, the accumulation of anthocyanin pigment can be 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 95% by mass or more, 99% by mass or more, or about 100% by mass relative to the total amount of pigment present in the cells. Furthermore, the accumulation of anthocyanin pigment and the total amount of pigment present in the cells can be determined, for example, by extracting the pigment from freeze-dried petals with a mixture of trifluoroacetic acid and acetonitrile and performing HPLC analysis. More specifically, see Examples 1 and 2 described below. In addition, there is no particular limit to the upper limit of the accumulation of anthocyanidin pigments, for example, it can be 100% by mass, 99.9% by mass or 99% by mass relative to the total amount of pigments present in the cells.

[0105] Furthermore, in this invention, there is no particular limitation on the amount of anthocyanin pigment in the plant after the accumulation of anthocyanin pigment. For example, it can be 0.05 mg or more, 0.1 mg or more, 0.2 mg or more, 0.3 mg or more, 0.4 mg or more, 0.5 mg or more, 0.6 mg or more, 0.7 mg or more, 0.8 mg or more, 0.9 mg or more, 1.0 mg or more, 1.0 mg or more, 1.2 mg or more, 1.3 mg or more, 1.4 mg or more, 1.5 mg or more, or 2.0 mg or more per 1g of petals. Alternatively, it can be less than 2.5 mg, less than 2.0 mg, less than 1.8 mg, less than 1.5 mg, or less than 1.1 mg.

[0106] In this invention, it is possible to conform to CIE Color system The CIE value is used to determine the change in flower color of plants before and after anthocyanin pigment accumulation. More specifically, it is defined as the change in flower color of plants after anthocyanin pigment accumulation compared to the flower color of plants before anthocyanin pigment accumulation. Color system The way the value is reduced can be changed.

[0107] Among them, CIE The color system is a standardized color system developed by the International Commission on Illumination (CIE). It is widely known as a method of describing hue and is also referred to as CIE 1976. (Color system) In the color system, using Indicate brightness, then use and This represents the hue and saturation of the color. and These represent the directions of the colors. Indicates the red direction, - Indicates the green direction. Indicates the yellow direction, and - Indicates the direction of cyan. , and The values ​​of each can be obtained from the tristimulus values ​​X, Y, and Z according to the following equation.

[0108] =116 (Y / Y0) 1 / 3 -16;

[0109] =500[(X / X0] 1 / 3 -(Y / Y0) 1 / 3 ];as well as

[0110] =200[(Y / Y0] 1 / 3 -(Z / Z0) 1 / 3 ]

[0111] Where X / X0, Y / Y0, and Z / Z0 are greater than 0.008856, and in the formula, X0, Y0, and Z0 represent the tristimulus values ​​of the standard light source.

[0112] Additionally, based on CIE Color analysis of color systems can be easily performed using an integrating sphere spectrophotometer, such as a commercially available spectrophotometer (CM-2022, Konica Minolta).

[0113] According to the method of the present invention, the A decrease in the value means that the flower color of plants that have accumulated anthocyanin pigments shifts towards a more bluish hue and changes accordingly. Here, for... There is no particular limitation on the degree of reduction of the value, for example, it can be 5.0 or above, 10.0 or above, 15.0 or above, 20.0 or above, 25.0 or above, 30.0 or above, 35.0 or above, 40.0 or above, 45.0 or above, or 50.0 or above.

[0114] Furthermore, in this invention, the flower color of plants after anthocyanin pigment accumulation is improved. The value is not specifically limited; for example, it can be below 20.0, below 15.0, below 10.0, below 5.0, below 1.0, or below 0. Additionally, The value of is preferably less than 0. Additionally, in When the value is less than 0, more specifically, its absolute value can be, for example, 1.0 or higher, 5.0 or higher, 10.0 or higher, 15.0 or higher, 20.0 or higher, 25.0 or higher, or 30.0 or higher.

[0115] In one embodiment, according to the method of the present invention, red-flowered Mandevilla (… Mandevilla In the case of plants belonging to the genus *Mandevilla*, compared with *Mandevilla stenoptera* before the accumulation of anthocyanin pigments (… Mandevilla Compared to the flower color (red) of plants in the genus *Mandevilla*, which accumulates anthocyanin pigments, the color of *Mandevilla* is significantly different. Mandevilla The flower color of plants in the genus () can be measured in CIE Color system The value changes as it decreases from "20.9" to "-30.9", meaning it can shift towards the cyan side.

[0116] The plant that can be applied to the method of the present invention is not particularly limited, and for example may be at least one selected from the Apocynaceae family ( Apocynaceae ) plants, Solanaceae ( Solanaceae ) plants, motherwort family ( Linderniaceae Plants and Scrophulariaceae ( Scrophulariaceae )plant.

[0117] Furthermore, the plant that can be applied to the method of the present invention can be selected from Mandevilla (… Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia It includes at least one of the genus *( )*, but is not limited thereto.

[0118] Generally, the red, blue, and purple flower colors found in plants are known to originate from anthocyanin pigments. Among anthocyanin aglycones, those with added sugars through glycosylation enzymes are anthocyanin pigments.

[0119] In this invention, the anthocyanidin aglycone is the aglycone of anthocyanin pigment and has the structure of a compound represented by the following general formula (I):

[0120]

Chemistry 1

[0121] (I)

[0122] (In equation (I), R) 1 R 2 R 3 R 4 R 5 R 6 and R 7 It can be an independent hydrogen atom, an -OH group, or an -OCH3 group.

[0123] In general formula (I), based on the number of hydroxyl groups in the B ring of the anthocyanidin moiety, it is divided into three systems: geranium pigment, cyanidin, and delphinidin. Furthermore, various anthocyanin pigments can exist based on the type and number of sugars bonded to the A or C ring of the anthocyanidin moiety.

[0124] In the method of the present invention, the anthocyanidin pigment may be cyanidin. Cyanidin has the structure shown in formula (II) below.

[0125]

Chemistry 2

[0126] (II)

[0127] In the method of the present invention, for example, a red-flowered Mandevilla vine ( Mandevilla In the case of plants belonging to the genus *Mandevilla*, cyanidin is preferably accumulated in the cells of the petals. Therefore, compared to *Mandevilla stenoptera* before cyanidin accumulation... Mandevilla Compared to the flower color (red) of plants in the genus *Mandevilla*, which accumulates cyanidin, the color of *Mandevilla* (*Mandevilla spp.*) is more pronounced. Mandevilla The flower color of plants in the genus 10 was changed to blue.

[0128] In this invention, anthocyanin pigments can be present locally in the cytoplasm of the cells of plant petals, particularly in the cytoplasm of the epidermal cells of plant petals. In contrast, anthocyanin pigments are generally present locally in the vacuoles of the cells of petals. Furthermore, the location of the pigment's local presence within the cell (i.e., the site of its presence) can be confirmed, for example, by preparing protoplasts from the petals and observing them using a phase-contrast microscope on a slide used for blood cell counting.

[0129] In the method of this invention, anthocyanin pigments are accumulated in the cells of plant petals by deleting at least a portion of the anthocyanin-modifying enzyme gene. Here, the anthocyanin-modifying enzyme can be an enzyme capable of modifying anthocyanin in its aglycone state, such as an enzyme for glycosylation or acylation of anthocyanin.

[0130] In one embodiment of the invention, the anthocyanidin modifying enzyme may be selected from anthocyanidin glycosylation enzyme and / or anthocyanidin 3-glucosidase glycosylation enzyme. In this case, in the method of the invention, anthocyanidin pigment accumulation can be achieved by deleting at least a portion of the anthocyanidin glycosylation enzyme gene and / or the anthocyanidin 3-glucosidase gene, preferably by deleting at least a portion of both the anthocyanidin glycosylation enzyme gene and the anthocyanidin 3-glucosidase gene.

[0131] While not theoretically limited, in the cells of plant petals, for example, by partially deleting the anthocyanin glycosylation enzyme gene and the anthocyanin 3-glucosidase gene, the anthocyanin pigment cannot be converted into its glycoside, i.e., anthocyanin, resulting in an accumulation of anthocyanin pigment. Furthermore, the deletion sites of the anthocyanin glycosylation enzyme gene and the anthocyanin 3-glucosidase gene are not particularly limited; for example, they can be their respective promoter regions.

[0132] Furthermore, the deletion of anthocyanidin-modifying enzyme genes can be achieved using conventional techniques in the art, such as mutagenesis induced by irradiation with gamma rays or heavy ion beams, RNAi, and genome editing. More specifically, this can be achieved, for example, by deleting, inserting, substituting, and / or adding at least a portion of the DNA sequence in the anthocyanidin-modifying enzyme gene.

[0133] In the method of the present invention, the anthocyanin glycosylation enzyme, specifically, may be a galactosyltransferase, for example. The gene for the galactosyltransferase may have the DNA sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 5. Alternatively, the anthocyanin 3-glucosidase glycosylation enzyme, specifically, may be a xylose transferase, for example. The gene for the xylose transferase may have the DNA sequence shown in SEQ ID NO: 6 and the amino acid sequence shown in SEQ ID NO: 7.

[0134] For example, in the presence of galactosyltransferase and xylose transferase, cyanidin can be converted into cyanidin glycoside as shown in formula (III) via a two-stage saccharification reaction. In the first stage, cyanidin is galactose-added at its 3-position by galactosyltransferase, becoming cyanidin 3-galactoside. Then, in the second stage, cyanidin 3-galactoside can be further galactose-added by xylose transferase.

[0135]

Transformation 3

[0136]

[0137] cyanidin 3-O-[2-O-(xylosyl)-galactoside] (III)

[0138] (In formula (III), Gal represents galactose and Xyl represents xylose.)

[0139] In addition, in the method of the present invention, when the anthocyanidin glycosylase is a galactosyltransferase, at least one sequence selected from the following can be deleted from the promoter region of the galactosyltransferase gene: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them.

[0140] Furthermore, to identify whether a sequence deletion exists in the galactosyltransferase gene, PCR primers can be designed to span the deleted portion, for example, by comparing it to genes with no sequence deletion. Then, PCR is performed using the designed primers; if an amplified fragment of the desired length is obtained, it can be identified as a sequence deletion.

[0141] In the method of the present invention, when the anthocyanidin 3-glucosidase is a xylose transferase, at least one sequence selected from the following can be inserted into the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity with it.

[0142] Furthermore, to identify whether a sequence insertion has occurred in xylose transferase, PCR primers can be designed from the inserted portion, for example, by comparing it with the case of a gene without a sequence insertion. Then, PCR is performed using the designed primers; if an amplified fragment of the desired length is obtained, it can be identified as a sequence insertion.

[0143] It should be noted that, in this invention, the term "identity" refers to the quantity (number) of the mutual fit between the amino acid residues or bases constituting the two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence) that can be determined to be the same. It means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. "Identity" can be easily calculated. There are many known methods for determining the identity between two polynucleotide sequences or polypeptide sequences, and the term “identity” is well known to those skilled in the art (see, for example, Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, AM & Grifin, HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.).

[0144] Furthermore, unless otherwise specified, the numerical value of "identity" described in this specification may be a value calculated using an identity retrieval program known to those skilled in the art, preferably a value calculated using the ClustalW program of the MacVector application (version 9.5 Oxford Molecular Ltd., Oxford, England). In this invention, the degree of "identity" between sequences may be, for example, about 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, more preferably 96% or more, further preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0145] In this invention, chlorogenic acid and aluminum ions can be present in the cells of plant petals. The presence of chlorogenic acid and aluminum ions allows for a stronger change in flower color due to their co-pigmenting effect. For example, when using Mandevilla chinensis (a plant with red flowers)... Mandevilla In the case of plants belonging to the genus *Mandevilla*, if chlorogenic acid and aluminum ions are present in the cells of the plant's petals, then they are associated with the accumulation of cyanidin in *Mandevilla stenoptera*. Mandevilla Compared to the flower color (red) of plants in the genus Mandevilla, which accumulates cyanidin, this can lead to a higher concentration of these pigments. Mandevilla The flower color of plants in the genus ) changes more strongly to blue.

[0146]

plant

[0147] This invention also provides a plant. The plant of this invention is the following:

[0148] In the cells of plant petals,

[0149] Anthocyanin pigments accumulate at a rate of over 30% by mass relative to the total amount of pigments present in cells, and

[0150] At least a portion of the anthocyanidin-modifying enzyme gene is missing.

[0151] The plant of the present invention can be produced by the method of the present invention described above. Therefore, the description of the anthocyanin-modifying enzyme can be appropriately referred to in the description of the method of the present invention described above.

[0152] Furthermore, in the plant of the present invention, at least a portion of the promoter region of the anthocyanidin modifying enzyme gene may be deleted. The anthocyanidin modifying enzyme may be selected from: anthocyanidin glycosylation enzyme and / or anthocyanidin 3-glucosidase. Additionally, when the anthocyanidin glycosylation enzyme is a galactosyltransferase, at least one sequence selected from the following may be deleted in the promoter region of the galactosyltransferase gene: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them. Furthermore, when the anthocyanidin 3-glucosidase is a xylose transferase, at least one sequence selected from the following may be inserted in the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3, and a sequence having at least 90% identity with it.

[0153] The anthocyanidin pigment of the plant of the present invention can be locally present in the cytoplasm of the cells of the plant petals. Alternatively, in the plant of the present invention, the anthocyanidin pigment can be cyanidin.

[0154] The plant of this invention may be selected from at least one of the following: Apocynaceae, Solanaceae, Hedyotis diffusa, and Scrophulariaceae plants; more specifically, it may be selected from at least one of the following: Mandevilla chinensis (… Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia Plants of the genus ().

[0155] The present invention relates to Mandevilla ( Mandevilla Plants of the genus *Mandevilla* can be obtained from *Mandevilla stenoptera* (with accession number FERM ABP-22483) deposited at the Patent Biological Collection Center (NITE-IPOD) with accession number FERM ABP-22483 using conventional techniques in plant varietal improvement. Mandevilla This invention relates to the production of *Mandevilla spp.* or its offspring. Conventional techniques for this purpose, besides existing propagation methods such as vegetative propagation and seed propagation, may include tissue culture techniques for culturing plant cells, tissues, or organs, and gene recombination techniques capable of directly introducing useful traits. The *Mandevilla spp.* involved in this invention... Mandevilla Plants of the genus *Mandevilla* are typically deposited on September 22, 2023, with accession number FERM ABP-22483 at the Patent Biological Collection Center (NITE-IPOD) of the Technical Base for Product Evaluation (NITE-IPOD) (accession number: FERM BP-22483; pursuant to the Budapest Treaty). Mandevilla Propagation is the vegetative propagation of plants of the genus ( ) or their offspring. In addition to methods such as cuttings (including leaf cuttings), grafting, layering, division, and grafting, cloning seedlings produced through tissue culture can also be used for "vegetative propagation," but cuttings are particularly preferred.

[0156] "Cutting" is a method of asexual reproduction, which involves cutting off a portion of a plant that lacks stems and roots to create an independent plant with stems and roots. Depending on the part used, it can be categorized into stem cuttings, root cuttings, leaf cuttings, etc. Typically, cuttings are performed by inserting a branch or part of the stem of the parent plant into the soil, leaving a portion exposed above ground. In this invention, there are no particular limitations on the part used for "cuttings".

[0157] Additionally, in propagation by cuttings, *Mandevilla stenoptera*, with accession number FERM ABP-22483, can also be used, deposited at the Patent Biological Collection Center (NITE-IPOD) of the Technical Base for Product Evaluation, an independent administrative agency, with accession number FERM ABP-22483. Mandevilla Varieties of plants of the genus *Mandevilla* or their offspring. A "variety" refers to a phenomenon in which, due to mutations in the growth point, the flower, new bud, or leaf of a particular branch of a plant exhibits a different genetic trait than that of the original individual, or represents an individual exhibiting this trait. In plants, because the plant body is continuously produced from the growth point forward, it is possible to distinguish it from the unmutated parts, thus establishing a fixed trait. Therefore, by using cuttings of varieties with at least one altered flower, it is possible to maintain a desired characteristic (e.g., blue flower color) and create mandevilla vines with other characteristics. Mandevilla A new variety of plant belonging to the genus ().

[0158] In another aspect of the invention, the invention provides the above-mentioned fragrant vine ( Mandevilla Propagation material (such as cuttings, seeds, etc.) of plants of the genus 10 or their offspring, a part of the plant body (flowers, stems, branches, leaves, roots, etc.), tissues, or cells.

[0159] In another embodiment of the invention, the invention provides a solution derived from the aforementioned Mandevilla vine ( Mandevilla Products of plants of the genus ( ) or their offspring, typically provided as potted plants, cut flowers, or processed products made from such cut flowers.

[0160] It should be noted that in this specification, "descendants" includes *Mandevilla stenoptera* with accession number FERM BP-22483, deposited at the Patent Biological Collection Center (NITE-IPOD), an independent administrative agency for the evaluation of products technology. Mandevilla The self-reproductive or other-reproductive offspring of plants in the genus *Mandevilla*, including not only *Mandevilla stenoptera* with accession number FERM ABP-22483 deposited at the Patent Biological Collection Center (NITE-IPOD), an independent administrative agency, but also those with accession number FERMBP-22483. Mandevilla The term "progeny" in this application specification refers to the first generation of offspring of plants in the genus *Mandevilla*, and also includes all generations of offspring. Furthermore, the term "progeny" in this application specification can include offspring derived from the parent plant *Mandevilla stenoptera* using conventional techniques in plant varietal improvement. Mandevilla Offspring produced from plants, for example, in addition to conventional propagation techniques such as vegetative propagation and seed propagation of the parent plant, also include offspring produced through tissue culture techniques that cultivate plant cells, tissues, or organs, gene recombination techniques that can directly introduce useful traits, or offspring produced from the parent plant Mandevilla (… MandevillaMutants such as bud mutations in plants. Such offspring are preferably derived from the parent plant, Mandevilla (…). Mandevilla The desired characteristics of plants of the genus ( ).

[0161]

Example

[0162] The present invention will be described in more detail below by way of examples, but the present invention is not limited thereto.

[0163]

Example 1

[0164] In Example 1, the blue-flowered variety (MW65) of Mandevilla vine (Plectranthus praecox) held by Suntory Flower Industry Co., Ltd. was used. Mandevilla The red variety (173) of the Mandevilla vine (with red flowers) x ), and their fragrant vines ( Mandevilla The pigment composition of the petal cells of the hybrid (13918) was analyzed by HPLC. Here, hybrid (13918) was obtained by crossing the green variety (MW65) as the female parent and a different phylogenetic variety from the red variety (173) as the male parent.

[0165] It should be noted that the above varieties (green varieties, red varieties, and their hybrids) adopt CIE standards. The color analysis of the color system was performed using a commercially available spectrophotometer (CM-2022, Konica Minolta). The results are shown in Table 1 below.

[0166] Table 1

[0167]

[0168] Non-patent literature 1 reports on the use of Mandevilla ( Mandevilla In the petal cells of plants in the genus *C.*, cyanidin is the main anthocyanin. Therefore, the anthocyanin pigments in the petals of each of the above varieties (blue variety, red variety, and their hybrids) were analyzed as follows.

[0169] [Preparation of test solution]

[0170] Add 4 mL of 0.1% TFA / 50% acetonitrile to 0.5 mg of each freeze-dried petal, and extract anthocyanin pigments under ultrasonic conditions for 20 minutes. Glycoside analysis was performed directly from the extract using HPLC. To remove aglycones such as sugars, the extract was dried overnight in a desiccator, dissolved in 6N HCl, and saponified by heating in boiling water for 20 minutes. After extraction with n-pentanol, the extract was analyzed by HPLC.

[0171] HPLC Analysis

[0172] As analytical instruments, Shimadzu Prominence HPLC systems (LC-20AD, SIL-20AC, CBM-20A, CTO-20AC, SPD-M20A, SPD-20A) were used. For glycoside analysis, an RSpak DE-413L column (250 mm × 4.6 mm ID, Shodex) was used as the mobile phase transport solution for the LC. Mobile phase A consisted of 0.5% TFA / water, and mobile phase B consisted of 50% CH3CN / water containing 0.5% TFA. A binary gradient was used, changing the concentration of mobile phase B from 20% to 100% over 15 minutes. Analysis was then performed at a flow rate of 0.4 mL / min under the condition of 100% mobile phase B concentration. For aglycone analysis, YMC-Pack ODS-A (150 mm × 6 mm ID, YMC) was used as the transport solution for the LC mobile phase, and acetic acid / methanol / water (15 / 17.5 / 67.5, vol / vol) was used at a flow rate of 1.0 mL / min. Peak identification and quantification were performed using cyanidin, delphinidin, geranium pigment, malva pigment, peony pigment, and petunia pigment as standards.

[0173] The result, such as Mandevilla As shown, in samples where sugars were removed by acid hydrolysis, cyanidin was found to be the main anthocyanin in both the green variety (MW65) and the red variety (173). Furthermore, this result is consistent with the report in Non-Patent Literature 1.

[0174] In addition, such as Figure 1 As shown, when analyzed in the presence of sugar, highly specific peaks (peak A and peak B) were detected in the red variety (173) and the green variety (MW65), respectively. In the hybrid variety (13918), two peaks (peak A and peak B) were detected. Then, in Example 2 shown below, the structure of these peaks was analyzed.

[0175]

Example 2

[0176] Using LC-MS, the structures of peaks A and B in the red variety (173) and green variety (MW65) of Example 1 were deduced.

[0177] More specifically, petal extracts were prepared using 5% AcOH, 50% MeCN-5% AcOH, and MeCN-5% AcOH. In LC-MS experiments, an LCMS-IT-TOF (Shimadzu) column was used, and a YMC-Triart C18 (3µm, 2.1×100mm, YMC) was used as the mobile phase delivery solution. Mobile phase A was set to 0.1% formic acid / water, and mobile phase B was set to 0.1% formic acid / acetonitrile. A binary gradient was used to change the concentration of mobile phase B from 20% to 100% over 15 minutes. Analysis was then performed at 100% mobile phase B concentration for 10 minutes at a flow rate of 0.4 mL / min. Anthocyanin peaks were extracted from the chromatogram at 530 nm, and structural analysis was performed based on MS and MS / MS spectra.

[0178] The result, such as Figure 2 As shown, peak A of the red variety (173) was found to be cyanidin 3-O-[2-O-(xylosyl)-galactoside], as described in Non-Patent Literature 1.

[0179] On the other hand, surprisingly, peak B of the blue variety (MW65) was found to be completely unmodified cyanidin (aglycone) = anthocyanin (aglycone) ( Figure 3 The analysis revealed that there was virtually no precedent for the high accumulation of aglycones in plants, a finding that overturns conventional wisdom.

[0180] In addition, the accumulation of anthocyanidin in the pericarp of common beans, depending on the variety, has been reported in Non-Patent Literature 2, but the proportion is 22%. In contrast, it was found that cyanidin accumulation, as shown by peak B, was over 70% by mass relative to the total amount of pigment in the cytoplasm and vacuoles of the petal cells of the green variety (MW65).

[0181]

Example 3

[0182] It is believed that in the Qing variety (MW65), anthocyanin is highly accumulated due to reduced expression or enzyme activity of the galactosyltransferase gene and xylose transferase gene, which are believed to be required for the synthesis of cyanidin 3-O-[2-O-(xylosyl)-galactoside] from cyanidin aglycone.

[0183] Therefore, firstly, the following identification was made of Mandevilla ( Figure 3 The galactosyltransferase gene and xylose transferase gene are found in *Mandevilla sylvestris*. Mandevilla From the gene population deduced from the genome sequence, glycotransferase genes were listed. The amino acid sequences of the proteins they encode were compared with the sequences of reported anthocyanin glycotransferases using ClustalW, and a phylogenetic tree was constructed using ETE3. From the obtained phylogenetic tree, genes considered to be orthologs of galactosyltransferase and xylose transferase genes from other reported plants were deduced. Expression analysis of this gene population was performed using RNA-seq and RT-PCR.

[0184]

RNA-seq method

[0185] Fruit-mate for RNA Purification (Takara Bio) was used to analyze RNA from Mandevilla rotundifolia (Plectranthus amboinicus). Mandevilla After pretreatment of the RNA from the flower petals, total RNA was extracted using RNeasy Plant mini (Qiagen). The RNA-Seq library was modulated using the MGI Easy RNADirectional Library Prep Set (MGI). Paired-end sequence data of 150 bases in length were obtained using the MGI DNBSeq-G400RS. After removing low-quality and adapter sequences, the sequences were mapped to a reference sequence, and expression levels were calculated.

[0186] Quantitative RT-PCR method

[0187] cDNA was prepared by reverse transcription of the total RNA using ReverTra Ace (Toyobo). Quantitative PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) and StepOnePlus real-time PCR system (Applied Biosystems). Actin gene expression was calculated as an endogenous control.

[0188] The result, such as MandevillaAs shown, in the red variety (173), both genes (galactosyltransferase gene and xylose transferase gene) showed high expression, but in the green variety (MW65), their expression was basically not observed. Furthermore, this result indicates that it is not related to the fact that "in the green series of *Pistacia chinensis* (…)…" Figure 4 This contradicts the idea that "most of the cyanidin in cyanidins is not modified by sugars and exists in the form of aglycones".

[0189]

Example 4

[0190] Generally, aglycones are known to be unstable and stabilized intracellularly through modifications such as glycosylation and acylation. As a potential source of stabilization for the highly accumulated cyanidin aglycone, a copigmentation effect exists (see, for example, Non-Patent Literature 3). This copigmentation effect is well-known in hydrangeas, resulting from the combination of anthocyanins with substances such as flavonoids (which are almost colorless or pale yellow) and metal ions within the organism, thus increasing the blue hue. Substances that could potentially contribute to the production of blue flowers were investigated using LC-MS in the blue cultivar (MW65).

[0191]

LC-MS

[0192] Petal extracts were prepared using 5% AcOH, 50% MeCN-5% AcOH, and MeCN-5% AcOH. In LC-MS experiments, an LCMS-IT-TOF (Shimadzu) was used as the instrument. A YMC-Triart C18 column (3µm, 2.1×100mm, YMC) was used as the mobile phase delivery solution. Mobile phase A consisted of 0.1% formic acid / water, and mobile phase B consisted of 0.1% formic acid / acetonitrile. The concentration of mobile phase B was changed from 20% to 100% over 15 minutes, and then the flow was carried out at 100% mobile phase B for 10 minutes at a flow rate of 0.4 mL / min. In the chromatogram at 325 nm, a blue-green Mandevilla vine ([unclear text - likely a typo, should be left as is]) was detected around 6.3 minutes. Mandevilla The peak was large. The structure of this peak was analyzed based on MS and MS / MS spectra.

[0193] The result, such as Mandevilla As shown, chlorogenic acid (3-CQA) accumulates at a high concentration in the Qing variety (MW65). This suggests that chlorogenic acid (3-CQA) contributes to the blue coloration of the Qing variety (MW65).

[0194]

Example 5

[0195] Add 2g of the Qing variety of fragrant vine ( Figure 5The petals of the garlic were juiced using a garlic juicer, yielding a red juice with a pH of 3.7 (see reference). Mandevilla (a) Based on this result, it is suggested that cyanidin aglycone is red at low pH, and that for it to produce a cyan color, cyanidin aglycone needs to be locally present in the cytoplasm.

[0196] Moreover, various solvents were used to treat the green variety of Mandevilla vine ( Figure 6 When the petals of the flower were extracted with amphiphilic methanol, all the pigment was extracted into the methanol, the petals turned white, and all the pigment was extracted (see reference). Mandevilla (b)). It is believed that cyanidin aglycone and glycoside were extracted simultaneously, and the methanol containing the pigment turned blue.

[0197] On the other hand, in the extraction using the organic solvent hexane (hydrophobic), hexane is colorless and transparent, but the petals turn red (see reference). Figure 6 (c) It is believed that the aglycones of the fat-soluble components were extracted, but the glycosides remained, and the petals were red.

[0198]

Example 6

[0199] To observe color development under neutral conditions simulating a cytoplasmic environment, in vitro pigment reconstruction experiments were conducted. Cyanidin hydrochloride (ChromaDex) and 3-O-caffeoylquinic acid (Nagara Scientific) were dissolved in DMSO to a concentration of 10 mM. A cyanidin-only reconstruction experiment was conducted by adding the cyanidin hydrochloride solution to a 50 mM phosphate buffer (pH 4.4–9.3) to a concentration of 0.5 mM, mixing, and reacting at room temperature. A reconstruction experiment was also conducted in the presence of co-pigments and metal ions by adding the cyanidin hydrochloride solution and 3-O-caffeoylquinic acid solution to a 50 mM phosphate buffer (pH 4.4–9.3) to a concentration of 0.5 mM, then adding an aluminum ion solution (1 mM aluminum ammonium sulfate solution) to a concentration of 0.1 mM, mixing, and reacting at room temperature (see reference). Figure 6 (Table).

[0200] As a result, cyanidin aglycone was observed to turn blue on its own at neutral pH (see reference). Figure 7 This strongly suggests that cyanidin aglycone can be produced independently in the presence of cyanidin in the cytoplasm, even without the action of co-pigments and metal ions.

[0201]

Example 7

[0202] To construct a biomarker specific to the Qing variety (MW65), the pyrotransferase gene and xylose transferase gene, along with their surrounding base sequences, were analyzed. The results showed that in the Qing variety, two deletions specifically occurred in the promoter region of the pyrotransferase gene (16 bp and 30 bp), and a specific insertion of 173 bp occurred in the promoter region of the xylose transferase gene (see reference). Figure 7 and Figure 8 In addition, since two sequences were obtained from the father and mother of the red variety (173), they were named 173-h1 and 173-h2, respectively. Therefore, the galactosyltransferase gene of the red variety 173-h1 has the sequence shown in SEQ ID NO: 8, and the galactosyltransferase gene of the red variety 173-h2 has the sequence shown in SEQ ID NO: 9. In addition, the xylose transferase gene of the red variety 173-h1 has the sequence shown in SEQ ID NO: 10, and the xylose transferase gene of the red variety 173-h2 has the sequence shown in SEQ ID NO: 11.

[0203] PCR primers spanning the deleted portion of the galactosyltransferase gene were designed (g21571-F7 (SEQ ID NO: 12): ACTGGCCTCG CCATTAACG, g21571-R5 (SEQ ID NO: 13): GTAATTTAAT TAAGATGCGTAATTCTCTG) (refer to...) Figure 9 ).

[0204] In addition, PCR primers were designed for the xylose transferase gene within this insertion region (g19028-F7 (SEQ ID NO: 14): AGTGTCTCTT CCTTAGTTCC TG, g19028-R5 (SEQ ID NO: 15): CTGAATTTTA AGAGATCGTTCATAATACG) (refer to... Figure 10 ).

[0205] DNA extracted from leaves of the green variety (MW65), red variety (173), and hybrid (13918) using NucleoSpin Plant II (MACHEREY-NAGEL) was used as a template, and PCR was performed using the primers described above. PCR was performed using Tks gflex DNA polymerase (Takara Bio) at 30 cycles of 98°C for 15 seconds, 60°C for 20 seconds, and 68°C for 30 seconds.

[0206] As a result, a 122bp amplified fragment of the galactosyltransferase gene was obtained in the blue variety (MW65) and the hybrid (13918), while no amplification was observed in the red variety (173) (see reference). Figure 10 (a)). For the xylose transferase gene, a 121 bp amplified fragment was obtained in the green variety (MW65) and the hybrid (13918), while no amplification was observed in the red variety (173) (see reference). Figure 11 (b)

[0207] In this way, by using these primers to perform PCR, it is possible to investigate whether the amplified fragment of the target length is obtained, thereby making it easy to identify the Qing variety (MW65) and its offspring.

[0208]

Example 8

[0209] To confirm that the pigment is locally present within cells, protoplasts were prepared from petals of the blue variety (MW65), the red variety (173), and the hybrid variety (13918) and the location of the pigment was confirmed.

[0210] Protoplasts were prepared as follows: 0.1 g of a petal slice and 10 mL of enzyme solution (1% (w / v) cellulase "ONOZUKA" RS (Yakult Pharmaceutical Industry), 1% (w / v) Mecezyme (Yakult Pharmaceutical Industry), 180 mM KCl, 20 mM CaCl2, 20 mM MgCl2, pH 5.5) were added to a 100 mL Erlenmeyer flask and incubated at 30 °C with shaking at 40 mm amplitude and 50 cycles per minute for 90 minutes. The mixture was then transferred to a 50 mL round-bottom glass centrifuge tube and centrifuged at 100 × g (730 rpm) for 2 minutes to precipitate the protoplasts. The precipitate was gently resuspended in the remaining liquid using a pipette. 10 mL of 0.5 M mannitol was added to the suspension and the precipitate was resuspended. This was used as the sample for observation. For observation, the sample was placed on a hemocytometer slide and observed using a phase-contrast microscope BX40 (Olympus).

[0211] In the red variety (173), the red pigment is evenly distributed in the vacuoles (see reference). Figure 11 In contrast to (a) and (b), in the Qing variety (MW65), it was observed that not vacuoles but cytoplasm was stained blue, and blue granular structures were also scattered (see reference). Figure 12 (e) and (f)).

[0212] In the mated strain (13918), two states were observed: vacuoles were uniformly stained red, while bluish granular structures were observed locally present in the cytoplasm (see reference). Figure 12 Figure 12 (c) and (d)). This clarifies that cyanidin aglycones are not locally located in the vacuoles containing the glycosides, but rather locally located within the cytoplasm, with some forming granular structures. This localized presence of the pigment in the cytoplasm is a previously unknown phenomenon and is considered a characteristic trait of the MW65 variety and its offspring.

[0213] [Rule 26, 22.11.2024]

[0214] PCT / RO / 134 form

Claims

1. Methods of producing plants, among which, The method includes: accumulating anthocyanin pigments in the cells of the plant petals to a concentration of 30% by mass or more relative to the total amount of pigments present in the cells, and Compared to the flower color of the plant before the accumulation of the anthocyanin pigment, the flower color of the plant after the accumulation of the anthocyanin pigment is as follows (CIE) Color system The way the value decreases changes.

2. The method according to claim 1, wherein, The anthocyanin pigment accumulates by deleting at least a portion of the anthocyanin-modifying enzyme gene.

3. The method according to claim 2, wherein, The method includes: deleting at least a portion of the promoter region of the anthocyanin-modifying enzyme gene, and The anthocyanidin modifying enzyme is selected from anthocyanidin glycosylation enzyme and / or anthocyanidin 3-glucosidase.

4. The method according to claim 3, wherein, The anthocyanin glycosylation enzyme is a galactosyltransferase, and In the promoter region of the galactosyltransferase gene, at least one sequence selected from the following is deleted: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them.

5. The method according to claim 3, wherein, The anthocyanidin 3-glucosidase is a xylose transferase, and Insert at least one sequence selected from the following into the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity with it.

6. The method according to any one of claims 1 to 5, wherein, The anthocyanidin pigment is cyanidin.

7. The method according to any one of claims 1 to 6, wherein, The anthocyanidin pigment is locally present in the cytoplasm of the cells.

8. The method according to any one of claims 1 to 7, wherein, The cells of the petals of this plant contain chlorogenic acid and aluminum ions.

9. The method according to any one of claims 1 to 8, wherein, The plant is selected from at least one of the following: Apocynaceae ( Apocynaceae ) plants, Solanaceae ( Solanaceae ) plants, motherwort family ( Linderniaceae Plants and Scrophulariaceae ( Scrophulariaceae )plant.

10. The method according to any one of claims 1 to 9, wherein, The plant is selected from at least one of the following: Mandevilla ( Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia Plants of the genus ().

11. The method according to claim 10, wherein, The fragrant vine ( Mandevilla Plants of the genus FERMABP-22483 are plants or their descendants deposited at the Patent Biological Collection Center (NITE-IPOD) with accession number FERMABP-22483.

12. Plants, among which, In the cells of the petals of the plant, Anthocyanin pigments accumulate to more than 30% by mass relative to the total amount of pigments present in the cells, and At least a portion of the anthocyanidin-modifying enzyme gene is missing.

13. The plant according to claim 12, wherein, The anthocyanidin pigment is locally present in the cytoplasm of the cells.

14. The plant according to claim 12 or 13, wherein, The plant comprises: a gene for modifying anthocyanins that has at least a portion of its promoter region deleted, and The anthocyanidin modifying enzyme is selected from anthocyanidin glycosylation enzyme and / or anthocyanidin 3-glucosidase.

15. The plant according to claim 14, wherein, The anthocyanin glycosylation enzyme is a galactosyltransferase, and In the promoter region of the galactosyltransferase gene, at least one sequence selected from the following is deleted: the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and a sequence having at least 90% identity with them.

16. The plant according to claim 14, wherein, The anthocyanidin 3-glucosidase is a xylose transferase, and Insert at least one sequence selected from the following into the promoter region of the xylose transferase gene: the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity with it.

17. The plant according to any one of claims 12 to 16, wherein, The anthocyanidin pigment is cyanidin.

18. The plant according to any one of claims 12 to 17, wherein, The cells of the petals of this plant contain chlorogenic acid and aluminum ions.

19. The plant according to any one of claims 12 to 18, wherein, The plant is selected from at least one of the following: Apocynaceae, Solanaceae, Solanaceae, and Scrophulariaceae.

20. The plant according to any one of claims 12 to 19, wherein, The plant is selected from at least one of the following: Mandevilla ( Mandevilla ) genus plants, petunia ( Petunia Plants of the genus ) and butterfly grass ( Torenia Plants of the genus ().

21. The plant according to claim 20, wherein, The fragrant vine ( Mandevilla Plants of the genus FERMABP-22483 are plants or their descendants deposited at the Patent Biological Collection Center (NITE-IPOD) with accession number FERMABP-22483.

22. The offspring of the plant according to any one of claims 12 to 21.

23. The propagation material of the plant according to any one of claims 12 to 21.

24. A part of the plant body, tissue, or cell of the plant according to any one of claims 12 to 21.

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