RU2823815C2 - S-methylmethionine-rich plant brassica oleracea - Google Patents

Abstract

FIELD: chemistry; biotechnology.

SUBSTANCE: invention relates to biochemistry, particularly to a method for increasing production of S-methylmethionine in Brassica oleracea. Also disclosed is a method of producing S-methylmethionine.

EFFECT: invention enables to efficiently obtain S-methylmethionine.

9 cl, 4 tbl, 2 dwg, 4 ex

Description

Field of technology to which the invention relates

[0001]

The present invention relates to Brassica oleracea plants having a higher S-methylmethionine content than in normal Brassica oleracea plants and to a method for producing them.

Prerequisites for the creation of an invention

[0002]

The plants of the Brassicaceae family are plant species native to the Middle East and the Mediterranean coast, and the plants of the genus Brassica cover very important agricultural crops. In particular, Brassica oleracea ( Brassica oleracea L. ) is a very important plant species covering B. oleracea L. var. capitata (cabbage), Brassica oleracea L. var. italica (broccoli), Brassica oleracea L. var. botrytis (cauliflower), B. oleracea L. var. gemmifera (Brussels sprouts), B. oleracea L. var. gongyloides (kohlrabi), B. oleracea L. var. acephala (fodder kale) and B. oleracea L. var. alboglabra (Chinese cabbage) and other species.

[0003]

Of the Brassica genus of the Brassicaceae family, broccoli has seen a steady increase in consumption in Japan since the 1980s. The florets and stems of broccoli are used as food because broccoli is rich in vitamin B, vitamin C, vitamin A, and dietary fiber. It is also rich in carotene and iron, and contains substances such as sulforaphane, which is believed to have anti-cancer properties. Thus, broccoli is recognized as a vegetable with many health benefits.

[0004]

S-methylmethionine is a sulfur-containing amino acid that is also sometimes called vitamin U or "cabagin" because it was found in cabbage. It is a factor that prevents the development of tumors of the digestive tract and is present in vegetables in its free form.

[0005]

The content of S-methylmethionine in vegetables was investigated. According to the results of the analysis presented in the report, the content of S-methylmethionine in vegetable cropsUmbelliferae,Solanaceae And Liliaceaeranges from 1 to 4 mg% based on raw weight, and the content in vegetable crops of the familyBrassicaceaeis from 4 to 20 mg% on a raw weight basis. As concerns, in particular, vegetable crops of the familyBrassicaceae, The content of S-methylmethionine in garden horseradish, Chinese cabbage and kale is relatively low (2-4 mg%), while in cauliflower, broccoli, kohlrabi and wild mustard it reaches 10-20 mg% (Non-patent literature 1). The content in green tea is from 1 to 9 mg% based on dry weight.

[0006]

According to the report regarding the distribution of nutritional components in various parts of broccoli, the compact inflorescence florets contain a larger amount of S-methylmethionine, and it is larger than other parts such as stems, lower parts of the main stem, main petiole, leaves and roots; that is, 16.7 mg/100 g fresh weight (Non-Patent Literature 2).

[0007]

Patent Literature 1 discloses a method for introducing heterologous chromosomes into plants to increase the amount of amino acids and/or an amino acid-associated substance in the plant. Although S-methylmethionine is a type of amino acid, Patent Literature 1 does not disclose Brassicaceae plants with an increased content of S-methylmethionine.

List of reference documents

Patent literature

[0008]

Patent Literature 1: JP 2013-143920 A

Non-patent literature

[0009]

Non-patent literature 1: Achievement report of fiscal year 1988, Kozo Ohtsuki, the scientific reports of Kyoto Prefectural University (Natural science and living science) 40, 1989

Non-patent literature 2: "Analysis of vitamin C content, S-methylmethionine content, and polyphenol content in different parts of broccoli and influence on cell function," the fiscal year of 2015, Fukuoka Industrial Technology Center, Research report No. 25

The essence of the invention

Technical task

[0010]

The present invention provides Brassica oleracea plants with a high content of S-methylmethionine.

Solution to the problem

[0011]

The inventor of the present invention has carried out research to obtain a Brassica oleracea plant with a high content of S-methylmethionine. This has led to the creation of the present invention.

(1) Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g fresh weight (FW) or more.

(2) A Brassica oleracea plant having a genetic trait causing increased levels of S-methylmethionine in the plant, wherein such genetic trait is present in broccoli, representative seeds of which have been deposited under accession number: FERM BP-22352, or in the progeny of broccoli having a genetic trait causing increased levels of S-methylmethionine in the plant.

(3) A Brassica oleracea plant according to paragraph (1) or (2), which is selected from the group consisting of broccoli, cauliflower, Chinese cabbage, cabbage, Brussels sprouts, kohlrabi, fodder kale and a hybrid thereof.

(4) The Brassica oleracea plant according to paragraph (3), which is broccoli.

(5) A part of the Brassica oleracea plant in accordance with any of paragraphs (1) to (4).

(6) A seed of the plant Brassica oleracea according to any of paragraphs (1) to (4).

(7) A method for producing a Brassica oleracea plant comprising the step of cross-pollinating the Brassica oleracea plant according to any of paragraphs (1) to (4) with another Brassica oleracea plant.

(8) The method according to paragraph (7), wherein the Brassica oleracea plant contains S-methylmethionine in an amount of 48 mg/100 g FW or more.

(9) The method according to paragraph (7) or (8), which further comprises the step of obtaining doubled haploids from the progeny plants obtained in the cross-pollination step by means of anther culture or pollen culture.

(10) A Brassica oleracea plant, a seed of a plant or a part of a plant that can be obtained by a process for obtaining a Brassica oleracea plant that includes the step of cross-pollinating a Brassica oleracea plant according to any of paragraphs (1) to (4) with another Brassica oleracea plant.

(11) A Brassica oleracea plant, seed of the plant or part of the plant according to paragraph (10), containing S-methylmethionine in an amount of 48 mg/100 g FW or more.

(12) A method for producing a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more, which comprises:

the stage of cross-pollination of the Brassica oleracea plant;

a step of obtaining doubled haploids from the progeny plant obtained in the step of cross-pollination by means of anther culture or pollen culture; and

the step of selecting a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more from doubled haploids obtained in the step of obtaining doubled haploids.

(13) An S-methylmethionine-containing composition comprising a Brassica oleracea plant according to any of paragraphs (1) to (4), a part of the plant, or a processed product of the plant or part.

(14) A method for producing S-methylmethionine, comprising obtaining S-methylmethionine from a Brassica oleracea plant according to any of paragraphs (1) to (4), a part of the plant, or a processed product of the plant or part.

(15) The use of the Brassica oleracea plant according to any of paragraphs (1) to (4), the seed of the plant or the part of the plant to obtain a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more.

(16) Brassica oleracea plant with higher S-methylmethionine content than normal Brassica oleracea plant.

(17) Brassica oleracea plant with high S-methylmethionine content, represented by broccoli, deposited under accession number: FERM BP-22352.

[0012]

The present specification includes part or all of the content disclosed in the description and/or drawings of Japanese Patent Application No. 2018-131069, which is a priority document of the present application.

Beneficial effects of the invention

[0013]

The present invention provides a Brassica oleracea plant with a high content of S-methylmethionine.

[0014]

The Brassica oleracea plant according to the present invention can be cross-pollinated with another Brassica oleracea plant to produce a Brassica oleracea plant with a high S-methylmethionine content.

[0015]

The S-methylmethionine-containing composition of the present invention is useful for applications such as a pharmaceutical composition or a food or beverage composition.

[0016]

According to the method for producing S-methylmethionine of the present invention, natural S-methylmethionine can be obtained.

Brief description of the drawings

[0017]

[Fig. 1] Fig. 1 is a diagram showing the relationship between the S-methylmethionine content and the number of plants among 95 F2 progeny plants derived from a hybrid obtained by crossing the 4-1 line (SSC-BRO-17-002) having a high S-methylmethionine content with another line.

[Fig. 2] Fig. 2 is a diagram showing 95 F2 progeny plants derived from a hybrid obtained by crossing line 4-1 (SSC-BRO-17-002), which has a high S-methylmethionine content, with another line, in order of increasing S-methylmethionine content.

Description of embodiments

[0018]

< Brassica oleracea plants>

Brassica oleracea ( Brassica oleracea L.), belonging to the family Brassicaceae , encompasses plants classified as broccoli, cauliflower, Chinese cabbage, cabbage, Brussels sprouts, kohlrabi, fodder kale, plants that can cross-pollinate with any of such plants, and hybrid plants resulting from cross-pollination of such plants. The term " Brassica oleracea plants" means plants that belong to the above species.

[0019]

In the present invention, broccoli includes Brassica oleracea plants belonging to Brassica oleracea L. var. italica and their progeny. In the present invention, the "progeny" of Brassica oleracea plants includes Brassica oleracea plants according to the present invention with a high S-methylmethionine content and plants obtained by cross-pollination between Brassica oleracea plants according to the present invention and other Brassica oleracea plants that can cross-pollinate with them. For example, hybrid species that can be obtained by cross-pollination between broccoli plants with a high S-methylmethionine content according to the present invention and other Brassica oleracea plants that can cross-pollinate with them are included in the scope of broccoli "progeny". Also, plants obtained by fusing cells of Brassica oleracea plants with a high S-methylmethionine content according to the present invention and other Brassica oleracea plants whose cells can be fused with them, interspecific hybrid plants, etc. are included in the scope of "progeny".

[0020]

In the present invention, the method for quantitatively determining S-methylmethionine is not particularly limited as long as the content of S-methylmethionine in a plant can be determined reproducibly. Examples of methods that can be used include a quantitative evaluation method using amino acid fluorescence labeling of a plant extract in HPLC, an indirect quantitative evaluation method using gas chromatography, an analytical method involving directly injecting a plant extract into a lithium-based amino acid analyzer and directly separating and quantifying S-methylmethionine, and a method of resuspending a precipitate extracted from a dried plant sample in a buffer and performing an analysis using an amino acid analyzer.

[0021]

In the present invention, for example, a broccoli sample for quantitative determination of S-methylmethionine content is obtained during harvesting of broccoli. Specifically, a broccoli sample is obtained when the diameter of the head, also called the inflorescence formed from flower buds, reaches its average size; i.e., 10-15 cm. Preferably, the harvested part of broccoli, which is commonly called the head, is a compound inflorescence, or a combined inflorescence, having an apical inflorescence and a plurality of lateral inflorescences, commonly called florets.

[0022]

The first aspect of the present invention relates to a Brassica oleracea plant having a higher S-methylmethionine content than that of a normal Brassica oleracea plant. When the S-methylmethionine content in the plants is higher than that of a normal Brassica oleracea plant, the S-methylmethionine content per unit mass of the plants is higher than that of a normal Brassica oleracea plant. Although the parts of the plant that can be analyzed and compared for the content of S-methylmethionine are not particularly limited, compact inflorescence flowers, stems or leaves are preferable, compact inflorescence flowers or stems are more preferable, and compact inflorescence flowers are particularly preferable.

[0023]

A specific example of Brassica oleracea plants having a higher S-methylmethionine content than ordinary Brassica oleracea plants is preferably Brassica oleracea plants containing S-methylmethionine in an amount of 48 mg/100 g FW or more. The S-methylmethionine content is preferably analyzed at the time of harvesting. In the case of broccoli, the S-methylmethionine content is more preferably analyzed when the diameter of the head reaches 10 to 15 cm. In the case of broccoli branches (also known as mini broccoli), the S-methylmethionine content is preferably analyzed when the diameter of the head reaches 3 to 4 cm. The S-methylmethionine content is preferably the S-methylmethionine content of the inflorescence flowers, stems or leaves, more preferably the S-methylmethionine content of the inflorescence flowers or stems, and particularly preferably the S-methylmethionine content of the inflorescence flowers. When the Brassica oleracea plants are broccoli, in particular, the S-methylmethionine content is 48 mg/100 g FW or more, preferably in the inflorescence flowers, stems or leaves at the time of harvest, more preferably in the inflorescence flowers or stems at the time of harvest, and particularly preferably in the inflorescence flowers at the time of harvest.

[0024]

The content of S-methylmethionine in the Brassica oleracea plants of the present invention is more preferably 50 mg/100 g FW or more, 55 mg/100 g FW or more, 60 mg/100 g FW or more, 65 mg/100 g FW or more, or 70 mg/100 g FW or more. The content of S-methylmethionine in the Brassica oleracea plants of the present invention is usually 200 mg/100 g FW or less, such as 150 mg/100 g FW or less, or 130 mg/100 g FW or less.

[0025]

The Brassica oleracea plants of the present invention are more preferably Brassica oleracea plants characterized by a high S-methylmethionine content, represented by broccoli, the seeds of which have been internationally deposited under accession number FERM BP-22352 (broccoli line: SSC-BRO-17-002), or their progeny, particularly preferably Brassica oleracea plants characterized by a high S-methylmethionine content, represented by broccoli, the seeds of which have been internationally deposited under accession number FERM BP-22352, and most preferably broccoli, the seeds of which have been internationally deposited under accession number FERM BP-22352, or their progeny. In this broccoli line, the S-methylmethionine content is particularly high.

[0026]

One or more other preferred embodiments of the Brassica oleracea plant according to the present invention relate to a Brassica oleracea plant having a genetic trait causing an increased content of S-methylmethionine in the plant, wherein such a genetic trait is present in broccoli, representative seeds of which are deposited under accession number: FERM BP-22352, or in the progeny of broccoli having a genetic trait causing an increased content of S-methylmethionine in the plant. The embodiments described below confirmed that the genetic trait of broccoli, representative seeds of which are deposited under accession number FERM BP-22352, causing an increased content of S-methylmethionine in plants is an inheritable genetic trait.

[0027]

The term "a genetic trait causing an increased content of S-methylmethionine in a plant" as used in the present application refers to a genetic trait such that the content of S-methylmethionine in the plants, preferably in the flowers of the inflorescence, stems or leaves, more preferably in the flowers of the inflorescence or stems, and particularly preferably in the flowers of the inflorescence, is preferably 48 mg/100 g FW or more, 50 mg/100 g FW or more, 55 mg/100 g FW or more, 60 mg/100 g FW or more, 65 mg/100 g FW or more, or 70 mg/100 g FW or more, more preferably up to 200 mg/100 g FW or less, such as 150 mg/100 g FW or less, or 130 mg/100 g FW or less.

[0028]

When Brassica oleracea plants according to an embodiment of the present invention have a genetic trait causing an increased S-methylmethionine content in the plant, and the genetic trait is "present" in broccoli, representative seeds of which are deposited under accession number: FERM BP-22352, or in the progeny of broccoli having a genetic trait causing an increased S-methylmethionine content in the plant, the genetic trait is the same (or substantially the same) as the genetic trait causing an increased S-methylmethionine content in the broccoli plants or their progeny. More preferably, the genetic trait causing an increased content of S-methylmethionine in Brassica oleracea plants according to an embodiment of the present invention may be derived from broccoli, representative seeds of which are deposited under accession number FERM BP-22352, or from the progeny of broccoli having the genetic trait causing an increased content of S-methylmethionine in plants. A person skilled in the art can easily transfer the genetic trait causing an increased content of S-methylmethionine in broccoli plants, representative seeds of which are deposited under accession number FERM BP-22352, or their progeny having such a genetic trait, to other plants using appropriate methods without undue trial and error.

[0029]

The second aspect of the present invention relates to parts of Brassica oleracea plants according to the present invention. Examples of parts of Brassica oleracea plants according to the present invention include inflorescence flowers, leaves, stems, roots, flowers, cells and nucleic acids, with inflorescence flowers, stems or leaves being preferred, inflorescence flowers or stems being more preferred, and inflorescence flowers being particularly preferred. Alternatively, a mixture of several parts may be used.

[0030]

A third aspect of the present invention relates to seeds of Brassica oleracea plants according to the present invention. Examples of seeds include seeds of the broccoli line: SSC-BRO-17-002, which are internationally deposited under accession number FERM BP-22352.

[0031]

<Method for producing Brassica oleracea plants with high content of S-methylmethionine 1>

A fourth aspect of the present invention relates to a method for producing Brassica oleracea plants comprising the step of crossing Brassica oleracea plants according to the present invention with other Brassica oleracea plants.

[0032]

Other Brassica oleracea plants are not particularly limited, provided that such plants can produce seed progeny as a result of crossing with the Brassica oleracea plants according to the present invention.

[0033]

In the Brassica oleracea plants obtained by this method, the content of S-methylmethionine is preferably increased compared to other parent Brassica oleracea plants. It is particularly preferable that the Brassica oleracea plants obtained by this method contain S-methylmethionine in an amount of 48 mg/100 g FW or more. A more preferable range of the content of S-methylmethionine in the plants is described above with respect to the Brassica oleracea plants according to the present invention.

[0034]

The method may further comprise a step of selecting plants with a high S-methylmethionine content from the plants obtained in the crossing step. The method for quantitatively determining the S-methylmethionine content in plants for selection is described above in connection with Brassica oleracea plants according to the present invention.

[0035]

The method for producing Brassica oleracea plants having a high S-methylmethionine content according to the present invention more preferably comprises, in addition to the crossing step, a step of producing doubled haploids from the progeny plants obtained in the crossing step by anther culture or pollen culture. The doubled haploids can be produced by anther culture or pollen culture according to a conventional method. For example, the doubled haploids can be produced according to Palmer, CE et al., 1996, "In Vitro Haploid Production in Higher Plants," Vol. 3, Kluwer Academic Publishers, (ed.): Jain, SM, Sopory, SK, and Veilleux, RE, pp. 143 to 172.

[0036]

<Method for producing Brassica oleracea plants with high content of S-methylmethionine 2>

A fifth aspect of the present invention relates to a method for producing Brassica oleracea plants containing S-methylmethionine in an amount of 48 mg/100 g FW or more, comprising:

the stage of cross-pollination of Brassica oleracea plants;

a step of obtaining doubled haploids from the progeny plants obtained in the step of cross-pollination by means of anther culture or pollen culture; and

the stage of selection of Brassica oleracea plants containing S-methylmethionine in an amount of 48 mg/100 g FW or more, from doubled haploids obtained in the stage of obtaining doubled haploids.

[0037]

In Example 4, it was found that a plurality of recessive factors are associated with the ability to produce S-methylmethionine in Brassica oleracea plants. Accordingly, it is considered very difficult to obtain Brassica oleracea plants containing S-methylmethionine in an amount of 48 mg/100 g FW or more by a conventional breeding method comprising repeated crossing and selection. According to the method of the present aspect comprising a crossing step, a doubled haploid producing step and a selection step, Brassica oleracea plants containing S-methylmethionine in an amount of 48 mg/100 g FW or more can be effectively obtained.

[0038]

The stage of obtaining doubled haploids can be carried out according to the procedure described in the literature mentioned above, or the like.

[0039]

Specific examples of Brassica oleracea plants are those described for Brassica oleracea plants according to the present invention.

[0040]

The preferred range of S-methylmethionine content and other properties of Brassica oleracea plants obtained by this method are those described for Brassica oleracea plants according to the present invention.

[0041]

The method for quantitative determination of S-methylmethionine content in plants for selection is similar to that described for Brassica oleracea plants in accordance with the present invention.

[0042]

<S-methylmethionine-containing composition>

A sixth aspect of the present invention relates to an S-methylmethionine-containing composition which comprises Brassica oleracea plants according to the present invention, parts thereof or processed products of the plants or parts.

[0043]

The composition containing S-methylmethionine is preferably a composition for oral administration, such as a pharmaceutical composition or a food or beverage composition.

[0044]

The Brassica oleracea plants or parts thereof according to the present invention are as described above.

[0045]

Examples of processed products of plants or parts thereof include products obtained as a result of at least one processing of plants or parts thereof, selected from dehydration, grinding, extraction and pressing. Products obtained as a result of two or more types of processing are also included in the scope of processed products of plants or parts.

[0046]

The composition containing S-methylmethionine may contain other components in addition to plants, their parts or processed products of plants or their parts. Examples of other components include excipients, carriers and solvents (e.g. water) that are acceptable for oral administration, such as for pharmaceuticals and food or beverage products.

[0047]

<Method for obtaining S-methylmethionine>

The seventh aspect of the present invention relates to a method for producing S-methylmethionine, comprising obtaining S-methylmethionine from Brassica oleracea plants of the present invention, parts thereof, or from processed products of the plants or parts thereof.

[0048]

Specific examples of plants, plant parts and processed products are described above.

[0049]

"Obtaining S-methylmethionine" is not limited to obtaining S-methylmethionine as a separate component and generally includes obtaining relatively highly concentrated S-methylmethionine from plants, their parts or their processed products. In particular, S-methylmethionine may be in the form of a crudely purified product.

[0050]

S-methylmethionine can be obtained from plants, their parts or their processed products using any procedure such as column chromatography or solvent extraction, and the procedure is not limited to this.

[0051]

The obtained S-methylmethionine can be used for its intended purpose, for example, in a pharmaceutical composition or a food or beverage composition.

Examples

[0052]

The present invention is described in more detail with reference to the following examples and comparative examples, although these examples are not intended to limit the scope of the present invention.

[0053]

<Cultivation Method: Plant Sampling Method>

After sowing the seeds, the seedlings grown for transplantation in the honeycomb trays were transplanted to the field, the seedlings were grown according to conventional methods, harvested when their heads were suitable for harvesting, and the collected inflorescences were stored in a freezer.

[0054]

<Method of quantitative determination: Method of quantitative determination of S-methylmethionine>

The plant samples were homogenized using 2% phosphoric acid solution. The homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was collected as S-methylmethionine extract. S-methylmethionine contained in the supernatant was reacted with amino acid drug fluorescence labeling reagent (NBD-F: 4-fluoro-7-nitrobenzofurazan) at 60°C for 5 min to obtain a fluorescent derivative, and the resulting substance was designated as an analytical sample. The analytical sample was analyzed using HPLC (LC10-ADvp, manufactured by Shimadzu Corporation) equipped with ODS columns (Shiseido Co., Ltd.) and a fluorescence detector to quantify S-methylmethionine.

[0055]

The amount of S-methylmethionine is given as the mass (mg) of S-methylmethionine per 100 g of raw mass of the part being studied (flowers, inflorescences, stems or leaves).

[0056]

Comparative Example 1

In the spring of 2006, 30 existing parental lines and first-generation hybrid (F1) varieties of broccoli were cultivated, their inflorescences were collected, and then samples were taken. Table 1 shows the results of quantitative determination of S-methylmethionine. As a result of detailed analysis, it was found that the inflorescence flowers of the H line of the present invention contained S-methylmethionine in an amount of 32.8 mg/100 g FW, which was the highest.

[0057]

Table 1 Variety Supplier Analyzed part S-methylmethionine
(mg/100 g FW) Green Magic Sakata Seed Corporation Flowers of the inflorescence 10.2 Green Magic Sakata Seed Corporation Leaves 0.5 Gypsy Sakata Seed Corporation Flowers of the inflorescence 10.9 Green Belt Sakata Seed Corporation Flowers of the inflorescence 8.7 Patron Sakata Seed Corporation Flowers of the inflorescence 9.9 Marathon Sakata Seed Corporation Flowers of the inflorescence 6.4 Maximo Sakata Seed Corporation Flowers of the inflorescence 9.9 K3-081 Sakata Seed Corporation Flowers of the inflorescence 14.1 K3-084 Sakata Seed Corporation Flowers of the inflorescence 7.2 K4-087 Sakata Seed Corporation Flowers of the inflorescence 6.8 SK0-070 Sakata Seed Corporation Flowers of the inflorescence 10.6 SK0-070 Sakata Seed Corporation Leaves 0.8 Sachiyoshi Mikado Kyowa Seed Co., Ltd. Flowers of the inflorescence 10.0 Takamori Mikado Kyowa Seed Co., Ltd. Flowers of the inflorescence 13.9 Forest Takii & Co., Ltd. Flowers of the inflorescence 8.5 Subaru Brolead Co., Ltd. Flowers of the inflorescence 10.0 Middle-early green port Marutane Co., Ltd. Flowers of the inflorescence 11.4 North bell Watanabe Noji Co., Ltd. Flowers of the inflorescence 14.6 Salinas early The Musashino Seed Co., Ltd. Flowers of the inflorescence 16.8 OASIS807F1 Agriseeds Flowers of the inflorescence 9.0 Florette Asgrow Flowers of the inflorescence 17.8 Line A Sakata Seed Corporation Flowers of the inflorescence 12.2 Line B Sakata Seed Corporation Flowers of the inflorescence 20.9 Line C Sakata Seed Corporation Flowers of the inflorescence 5.6 Line D Sakata Seed Corporation Flowers of the inflorescence 11.6 Line E Sakata Seed Corporation Flowers of the inflorescence 10.9 Line 1 Sakata Seed Corporation Flowers of the inflorescence 7.9 Line F Sakata Seed Corporation Flowers of the inflorescence 12.6 G Line Sakata Seed Corporation Flowers of the inflorescence 28.5 Line H Sakata Seed Corporation Flowers of the inflorescence 32.8

[0058]

Example 1

The author of the present invention obtained the broccoli line 4-1 (SSC-BRO-17-002) from a doubled haploid line obtained by anther culture from the progeny obtained by crossing the hybrid progeny of broccoli line 1 and broccoli line 2 with broccoli line 3.

[0059]

Surprisingly, the present inventor found such properties that the content of S-methylmethionine was significantly high in SSC-BRO-17-002 and in a new F1 variety obtained by crossing SSC-BRO-17-002 with another broccoli line 5 (see Examples 2 and 3).

[0060]

Broccoli seed line SSC-BRO-17-002 was deposited internationally with the Patent Microorganism Depository, the National Institute of Technology and Evaluation (#120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on December 15, 2017 (Depositor ID: SSC-BRO-17-002; Accession No.: FERM BP-22352).

[0061]

Example 2

To determine the variation in S-methylmethionine content in broccoli plants, four broccoli varieties sown and transplanted between August and September 2012 in Field 1 or Field 2 were analyzed for S-methylmethionine content to observe seasonal variations. The results are shown in Table 2. The flowers of the inflorescences were analyzed. It was found that the new F1 hybrid variety described in Example 1 had very high S-methylmethionine content compared to other existing broccoli varieties.

[0062]

Table 2 Variety Place of cultivation Sowing date Date of sampling S-methylmethionine
(mg/100 g FW) Pixel
(Sakata Seed
Corporation) Field 1 August 1 October 24 17 August 1 October 25 13 August 10 November 8 21 August 20 January 7 30 Oh, ha, you
(Sakata Seed
Corporation) Field 1 August 1 October 22 17 August 1 October 24 17 August 10 October 31 18 August 20 January 17 18 August 30 February 5 12 Grandome
(Sakata Seed
Corporation) Field 1 August 1 November 28 15 August 1 November 28 21 August 10 December 13 26 August 20 February 5 2 August 30 February 21 14 September 10 February 28 7 September 20 March 15 17 Field 2 August 6 November 15 40 August 20 January 8 38 New variety F1 Field 1 August 1 November 8 63 August 1 November 8 49 August 10 November 20 72 August 20 February 5 87 August 30 February 21 54 Field 2 August 5 November 7 82 August 20 January 8 123

[0063]

Example 3

The broccoli lines shown in Table 3 were grown in Field 1 in 2015 and analyzed for S-methylmethionine content in the inflorescence flowers. The analyses were performed using inflorescences collected from 3 plants per line and average values were determined. The results are shown in Table 3.

[0064]

Line 5 and SSC-BRO-17-002 are as described in Example 1, and line G is as described in Comparative Example 1.

[0065]

As described in Example 1, line 4-1 (SSC-BRO-17-002) and lines 4-2, 4-3, 4-4, 4-5 and 4-6 shown in Table 3 were derived from doubled haploid lines obtained by anther culture from the progeny resulting from crossing the hybrid progeny of parental line 1 and parental line 2 with parental line 3.

[0066]

Line 7 was selected from the hybrid progeny of line 5 and line 8.

[0067]

As a result, it was found that the content of S-methylmethionine was 48 mg/100 g FW or more in lines 4-1, 4-3, 4-5 and 4-6 and line 7.

[0068]

Table 3 Line Harvest date S-methylmethionine
(mg/100 g FW) 1 2 3 Average value Line 5 12/27 36 31 40 36 Line 1 12/5 11 22 19 17 J Line 1/11 - 2/21 40 9 4 18 G Line 1/31 - 2/21 10 7 7 8 Line 6 12/27 9 14 13 12 Line 4-1 (SSC-BRO-17-002) 12/5 - 12/16 64 94 67 75 Line K 1/19 - 1/31 18 12 20 17 L Line 1/31 34 29 25 29 M Line 12/27 20 23 19 21 Line N 12/20 - 12/27 5 6 8 6 Q Line 12/20 8 10 10 9 Line P 12/20 7 9 12 9 Line 7 3/7 49 49 64 54 Line 4-2 12/9 28 41 53 41 Line 4-3 12/9 55 67 65 62 Line 4-4 12/20 5 4 7 5 Line 4-5 12/20 46 57 41 48 Line 4-6 1/11 46 55 50

[0069]

Example 4 (hereditary patterns)

Seeds of broccoli line 4-1 (SSC-BRO-17-002) obtained in Example 1, broccoli line 5 described in Example 1, a new F1 variety described in Example 1 as an F1 hybrid of line 4-1 and line 5, another broccoli line 6, F1-2 as an F1 hybrid of line 4-1 and line 6, and F1-2-1 as an F2 progeny self-reproduced from F1-2 were obtained and planted to investigate the hereditary patterns of S-methylmethionine content. Six parental line plants (line 4-1 (SSC-BRO-17-002), line 5 and line 6), 9 F1 hybrid plants (new F1 variety, F1-2) and 95 F2 progeny plants (F1-2-1) were analyzed to confirm the distribution of S-methylmethionine content in the inflorescence flowers and the mean values were determined. The F2 progeny plant population (F1-2-1) showed the distribution shown in Table 1. Table 4 shows the mean value of S-methylmethionine content for each line.

[0070]

As shown in Fig. 1, most of the 95 F2 progeny plants (F1-2-1) were found to have low S-methylmethionine content and a low average content (24 mg/100 g FW). Fig. 2 shows the results for the 95 F2 progeny plants (F1-2-1) in ascending order of S-methylmethionine content. The distribution status is shown from a minimum of 5 mg/100 g FW to a maximum of 103 mg/100 g FW. Based on the S-methylmethionine content of the F2 progeny plants of line 4-1 and line 6, analyzed independently, it can be inferred that multiple recessive factors are involved, although the details remain unknown.

[0071]

Table 4 Line S-methylmethionine
(mg/100 g FW) Line 5 37 Line 4-1 (SSC-BRO-17-002) 85 New F1 variety 51 Line 6 15 F1-2 20 F1-2-1 24

[0072]

All publications, patents and patent applications cited in this application are incorporated herein by reference in their entirety.

Claims (23)

1. A method for increasing the production of S-methylmethionine in the plant Brassica oleracea, comprising: a cross-pollination stage between a Brassica oleracea plant having a trait causing increased S-methylmethionine content and another Brassica oleracea plant, such trait being present in representative seeds deposited under accession number: FERM BP-22352; a step of obtaining doubled haploids from the progeny of the plant obtained in the step of cross-pollination by means of anther culture or pollen culture; and a step of selecting a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more from doubled haploids obtained by a step of obtaining doubled haploids. 2. A method for increasing the production of S-methylmethionine in the plant Brassica oleracea, comprising: the stage of cross-pollination of a Brassica oleracea plant, a representative seed of which is deposited under accession number: FERM BP-22352, and another Brassica oleracea plant; a step of obtaining doubled haploids from the progeny of the plant obtained in the step of cross-pollination by means of anther culture or pollen culture; and a step of selecting a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more from doubled haploids obtained by a step of obtaining doubled haploids. 3. The method according to claim 1, wherein the Brassica oleracea plant is selected from the group consisting of broccoli, cauliflower, Chinese cabbage, cabbage, Brussels sprouts, kohlrabi, fodder kale and their hybrids. 4. The method according to claim 1, wherein the Brassica oleracea plant is broccoli. 5. A method for producing S-methylmethionine, comprising: a cross-pollination stage between a Brassica oleracea plant having a trait causing increased S-methylmethionine content and another Brassica oleracea plant, such trait being present in representative seeds deposited under accession number: FERM BP-22352; the stage of obtaining doubled haploids from the progeny of a plant obtained at the stage of cross-pollination by means of anther culture or pollen culture; a step of selecting a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more from doubled haploids obtained by a step of obtaining doubled haploids; and obtaining S-methylmethionine from a previous stage Brassica oleracea plant or a processing product of the said plant or part thereof. 6. A method for producing S-methylmethionine, comprising: the stage of cross-pollination of a Brassica oleracea plant, a representative seed of which is deposited under accession number: FERM BP-22352, and another Brassica oleracea plant; the stage of obtaining doubled haploids from the progeny of a plant obtained at the stage of cross-pollination by means of anther culture or pollen culture; a step of selecting a Brassica oleracea plant containing S-methylmethionine in an amount of 48 mg/100 g FW or more from doubled haploids obtained by a step of obtaining doubled haploids; and obtaining S-methylmethionine from a previous stage Brassica oleracea plant or a processing product of the said plant or part thereof. 7. The method according to claim 5, wherein the Brassica oleracea plant is selected from the group consisting of broccoli, cauliflower, Chinese cabbage, cabbage, Brussels sprouts, kohlrabi, fodder kale and their hybrids. 8. The method according to claim 5, wherein the Brassica oleracea plant is broccoli. 9. The method according to claim 5, wherein the part is inflorescences.