Intergeneric hybrid plants of the Brassicaceae family with high glucoraphanin content and method for producing the same

Abstract

The present invention achieves a Brassica plant having a high content of glucoraphanin.　The present invention also achieves an intergeneric crossbred plant in which have been crossed a first parent plant and a second parent plant, which are Brassica plants classified into different genera. The first parent plant includes 5 mg / 100 g (fresh weight) or more of glucoraphanin. The second parent plant includes a loss-of-function type glucoraphasatin synthetase gene.

Description

[Technical Field]

[0001] The present invention relates to an intergeneric hybrid plant of the Brassicaceae family that is high in glucoraphanin, and a method for producing the same. [Background technology]

[0002] Sulforaphane, a type of isothiocyanate, is a phytochemical found in plants of the Brassicaceae family, particularly the Brassica oleracea species. Sulforaphane is a functional component known to have bioactivities such as cancer prevention by activating the production of detoxification enzymes in the human body, as well as liver function improvement and antioxidant effects (see, for example, Non-Patent Document 1).

[0003] Normally, sulforaphane exists in plant cells in the form of its precursor, glucoraphanin. Glucoraphanin is a type of secondary metabolite glucosinolate also known as mustard oil glycoside. Glucoraphanin in the cells is exposed outside the cells through chewing or other processes, and is converted to sulforaphane by reacting with the enzyme myrosinase present in the plant or by being decomposed by intestinal bacteria.

[0004] Patent Literature 1 discloses a method for obtaining Brassica species with high glucoraphanin content by crossing wild-type Brassica species with a Brassica oleracea breeding line and selecting hybrids with higher 4-methylsulfinylbutyl glucosinolate (glucoraphanin) levels than the original breeding line. Patent Literature 2 discloses Brassica oleracea plants with high glucosinolate content that contain a Myb28 allele from Brassica villosa and a deletion of the ELONG allele from Brassica villosa genetically linked to the Myb allele.

[0005] Raphanus species of the Brassicaceae family do not usually contain glucoraphanin at usable levels. Patent Document 3 discloses a method for producing radish lines with high glucoraphanin content by selecting individuals with high glucoerucin content and a 4-methylthio-3-butenyl glucosinolate (glucoraphasatin) content of 1 / 5 or less of the glucoerucin content, and then performing self-fertilization.

[0006] Non-Patent Document 2 reports that high contents of glucoraphanin and glucoraphenin were detected in Raphanobrassica, an intergeneric hybrid of radish (Raphanus genus) of the Brassicaceae family and kale (Brassica genus).

[0007] It is known that glucoraphasatin typically accounts for more than 90% of the glucosinolates contained in radishes. Non-Patent Document 3 reports the identification of the glucoraphasatin synthase (GRS) gene. Patent Document 4 also discloses a method for crossbreeding radish individuals having a defective GRS gene to obtain a radish line with a low glucoraphasatin content in order to reduce the distinctive odor and yellowing of radish that result from the degradation products of glucoraphasatin. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2002-511235 [Patent Document 2] JP 2014-76045 A [Patent Document 3] Japanese Patent Application Laid-Open No. 2012-110238 [Patent Document 4] Japanese Patent Application Laid-Open No. 2016-86761 [Non-patent literature]

[0009] [Non-Patent Document 1] Zhang, Y. et al., P. Proc. Natl. Acad. Sci., Vol.91, pp. 3147-3150 (1994) [Non-patent document 2] Nagano Prefectural Agricultural Experiment Station website, Research Information, Research Results "Technical Information" [Result Name] Raphanobrassica "Nagano No. 48" is a promising new vegetable containing many functional ingredients (URL https: / / www.agries-nagano.jp / wp / wp-content / uploads / 2019 / 04 / 2018-2-g10.pdf) [Non-patent document 3] Kakizaki, T. et. al., Plant Physiology, Vol. 173, pp. 1583-1593 (2017) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to obtain a Brassicaceae plant having a high glucoraphanin content. [Means for solving the problem]

[0011] In order to achieve the above-mentioned objective, the inventors conducted extensive research and discovered that intergeneric hybrids obtained by crossing a Brassica plant with a Radish plant having a functionally defective GRS gene have a high glucoraphanin content, thereby completing the present invention.

[0012] The present invention encompasses the following. (1) An intergeneric hybrid plant between a Brassica plant and a Radish plant, having a ratio of glucoraphanin content to glucoraphenin content of 1.0 or more. (2) The plant according to (1), having a glucoraphanin content of 20 mg / 100 g (fresh weight) or more. (3) A plant according to (1) or (2), having a glucoraphenin content of 50 mg / 100 g (fresh weight) or less. (4) An intergeneric hybrid plant between a Brassica plant and a Radish plant, which contains a functionally defective glucoraphasatin synthase gene. (5) The plant according to (4), wherein the functionally defective glucoraphasatin synthesis gene is the following gene (a) and / or (b): (a) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, in which the exon constituting the gene is missing all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein; (b) A gene containing a nucleotide sequence having 70% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 3. (6) The plant according to any one of (1) to (5), wherein the Brassica plant is Brassica oleracea. (7) The plant according to any one of (1) to (6), wherein the plant of the genus Raphanus is Raphanus sativus. (8) The plant according to any one of (1) to (7), which has a polyploid chromosome. (9) A method for producing an intergeneric hybrid plant of the Brassicaceae family, comprising the steps of crossing a first parent plant with a second parent plant and obtaining an intergeneric hybrid plant between the first parent plant and the second parent plant, wherein the first parent plant and the second parent plant are Brassicaceae plants but of different genera, the first parent plant contains glucoraphanin at a concentration of 5 mg / 100 g (fresh weight) or more, and the second parent plant contains a functionally defective glucoraphasatin synthase gene. (10) The method according to (9), wherein the functionally defective glucoraphasatin synthase gene is the following gene (a) and / or (b): (a) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, in which the exon constituting the gene is missing all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein; (b) A gene containing a nucleotide sequence having 70% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 3. (11) The method according to (9) or (10), further comprising a step of selecting, from the intergeneric hybrid plants, an intergeneric hybrid plant containing a functionally defective glucoraphasatin synthase gene. (12) The method according to any one of (9) to (11), wherein the first parent plant is a plant of the genus Brassica, and the second parent plant is a plant of the genus Radish. (13) The method according to any one of (9) to (12), wherein the Brassica plant is Brassica oleracea. (14) The method according to any one of (9) to (13), wherein the plant of the genus Raphanus is Raphanus sativus. (15) The method according to any one of (9) to (14), wherein the intergeneric hybrid plant contains a polyploid chromosome. (16) A method for producing a Brassicaceae plant, comprising the step of cultivating the plant according to any one of (1) to (7). (17) A food product made from a plant according to any one of (1) to (7). (18) A method for increasing the glucoraphanin content of a Brassicaceae plant, comprising: a first step of preparing a Brassicaceae plant containing 5 mg / 100 g (fresh weight) or more of glucoraphanin as a first parent plant; a second step of preparing a Brassicaceae plant of a different genus from the first parent plant as a second parent plant, which has a lack of or reduced function of glucoraphanin synthase; and a third step of crossing the first parent plant with the second parent plant. (19) The method according to (18), wherein the second step comprises modifying the glucoraphasatin synthase gene to eliminate or reduce the function of glucoraphasatin synthase. This specification includes the disclosure of Japanese Patent Application No. 2021-063196, from which this application claims priority. [Effects of the Invention]

[0013] According to the present invention, a Brassicaceae plant having a higher glucoraphanin content can be obtained. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the biosynthetic pathway of glucoraphanin and glucoraphenin in Brassicaceae plants. [Figure 2] Schematic diagrams showing the structure of the wild-type GRS1 gene and the retrotransposon insertion positions in the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. (A) shows the structure of the wild-type GRS1 gene. (B) shows the structure of the nucleotide sequence shown in SEQ ID NO: 2. (C) shows the structure of the nucleotide sequence shown in SEQ ID NO: 3. [Figure 3] 1 is a graph showing the correlation between the glucoraphanin content in leaves and seeds of individual Brassicaceae plants. DETAILED DESCRIPTION OF THE INVENTION

[0015] As used herein, the chemical formulas described include all geometric and optical isomers unless otherwise specified. As used herein, the term "content" refers to weight concentration (w / w) unless otherwise specified. As used herein, the term "derivative" refers to a compound that has been modified to such an extent that the skeleton structure of the compound is not affected.

[0016] 1. Intergeneric hybrid plants of Brassica and Radish A first embodiment of the present invention is an intergeneric hybrid plant of a Brassica plant and a Radish plant (hereinafter also referred to as "the plant of the present invention") characterized in that the ratio of glucoraphanin content to glucoraphenin content is 1.0 or more. A second embodiment of the present invention is characterized in that the plant contains a functionally defective glucoraphasatin synthase gene. The plant of the present invention may have either one or both of the features of the first and second embodiments.

[0017] In this specification, when simply referring to a "plant," it is intended to include any of the leaves, stems, flowers, buds, roots, and seeds unless otherwise specified, and unless there is a particular contradiction. The plant of the present invention has the above-mentioned characteristics in at least any of the leaves, stems, flowers, buds, roots, and seeds.

[0018] As used herein, the term "glucoraphanin" refers to a compound represented by the following formula (I), a derivative thereof, or a salt thereof. [ka]

[0019] As used herein, the term "glucoraphenin" refers to a compound represented by the following formula (II), a derivative thereof, or a salt thereof. [ka]

[0020] It has been revealed that glucoraphanin and glucoraphenin are biosynthesized in Brassicaceae plants via the biosynthetic pathway shown in Figure 1. Specifically, glucoerucin is synthesized using methionine as the starting substance through approximately 20 enzymatic reactions. Glucoraphanin is synthesized by oxidizing glucoerucin. In some Brassicaceae plants, glucoerucin is converted to glucoraphasatin by the glucoraphasatin synthase (GRS1) gene, and glucoraphasatin is then oxidized to synthesize glucoraphenin. Glucoraphasatin is converted to the pungent component laphasatin by the action of the hydrolytic enzyme myrosinase, and is then converted into odor-causing components and yellowing substances.

[0021] As used herein, "Brassicaceae plants" are plants classified into a family in the division of Angiosperms, class of Dicotyledons, subclass of Azotophytes, and order Capparadis, and include plants of many genera such as Cardamine, Brassica, Brassica, Carpinus, Radish, and Variegata.

[0022] As used herein, "Brassica plants" refer to plants classified as a genus in the Brassicaceae family, including rape, mizuna, taisai, bok choy, komatsuna, turnip, Chinese cabbage, cabbage, broccoli, snapdragon, mustard, kale, kohlrabi, and cauliflower. Brassica plants used as parent plants for the plants of the present invention are preferably plants containing relatively large amounts of glucoraphanin, such as kale, broccoli, cabbage, kohlrabi, and cauliflower. The Brassica plant is preferably Brassica oleracea. In particular, Brassica plants containing glucoraphanin at a concentration of 5 mg / 100 g (fresh weight (FW)) or more, or 10 mg / 100 g FW or more, are preferred. Such glucoraphanin-rich Brassica oleracea may be obtained, for example, by the method described in Patent Document 1.

[0023] As used herein, "plants of the genus Raphanus" refers to plants classified into a genus in the Brassicaceae family, and includes radish, wild radish, etc. The radish plant used as the parent plant of the plant of the present invention is preferably Raphanus sativus.

[0024] Radish plants, especially radish, are characterized by the presence of glucoraphasatin, a compound not synthesized in closely related species of the Brassicaceae family. The gene encoding glucoraphasatin synthase, which converts glucoerucin into glucoraphasatin, has been identified in radish, and it has been revealed that glucoraphasatin is synthesized by this unique gene.

[0025] As described in Patent Document 4 and elsewhere, some radish mutants have low glucoraphasatin content. It is known that in these mutants, the structure of the glucoraphasatin synthase gene present at the end of the first linkage group of radish has changed from the wild type, resulting in loss of its function. Because the normal glucoraphasatin synthase genotype is dominant, it was named the GRS1 (Glucoraphasatin Synthase 1) gene, while the recessive genotype that lost its function was named the grs1 gene. The amino acid sequence encoded by the GRS1 gene contains a 2-oxoglutarate-iron(II)-dependent oxygenase domain, an oxygenase commonly found in plants.

[0026] The radish plant used in the present invention is preferably a radish plant containing a functionally defective glucoraphasatin synthase (grs1) gene. The grs1 contained in the radish plant may be present as a homozygote or heterozygote. Radish plants containing a functionally defective glucoraphasatin synthase (grs1) gene can be selected, for example, using the DNA marker assay technique described in Patent Document 4. Specifically, a polymerase chain reaction (PCR) method can be used, using DNA extracted from a sample plant as a template, a primer set that specifically amplifies the GRS1 gene, and a primer set that specifically amplifies the grs1 gene.

[0027] For plants of the genus Radish containing the grs1 gene, mutants may be selected by the above-mentioned methods from a large number of progeny lines produced by outcrossing, or wild-type plants of the genus Radish may be used in which the GRS1 gene has been modified to eliminate or reduce its function. Any known method can be used to modify the GRS1 gene. Examples include mutagenesis involving the introduction of an insertion sequence via a transposon, retrotransposon, plant virus, or the like. Other examples include mutation treatments such as seed irradiation, heavy ion beam treatment, and treatment with a solution containing a mutagen.

[0028] As used herein, the term "intergeneric hybrid plant" refers to a hybrid formed by crossing organisms classified into different genera, i.e., a hybrid progeny. It is clearly distinguished from an interspecific hybrid, which is a hybrid between different organisms within the same genus.

[0029] The plant of the present invention is a hybrid progeny produced by crossing a Brassica plant with a Radish plant. In a first embodiment of the plant of the present invention, the ratio of glucoraphanin content to glucoraphenin content is 1.0 or greater. In this embodiment, the amount of glucoraphanin contained in the plant of the present invention is preferably 20 mg / 100 g FW or greater, particularly 30 mg / 100 g FW or greater, 50 mg / 100 g FW or greater, 100 mg / 100 g FW or greater, or 150 mg / 100 g FW or greater. Furthermore, the amount of glucoraphenin contained in the plant of the present invention is preferably 50 mg / 100 g FW or less, particularly 20 mg / 100 g FW or less, 10 mg / 100 g FW or less, 5 mg / 100 g FW or less, 3 mg / 100 g FW or less, or 2 mg / 100 g FW or less.

[0030] A second embodiment of the plant of the present invention comprises a loss-of-function glucoraphasatin synthase gene. In this embodiment, the plant of the present invention is produced by using a Radish plant that possesses a loss-of-function glucoraphasatin synthase gene, the grs1 gene, in either a homozygous or heterozygous form as a parent plant. When a Radish plant that possesses a heterozygous grs1 gene is used, half of the intergeneric hybrid plants will be loss-of-function. Therefore, the plant of the present invention can be obtained by selecting loss-of-function plants and obtaining progeny. A method for selecting loss-of-function plants can be, for example, the DNA marker assay described in Patent Document 4. Specifically, a PCR method can be used using DNA extracted from a sample plant as a template, a primer set that specifically amplifies the GRS1 gene, and a primer set that specifically amplifies the grs1 gene.

[0031] In this embodiment, the structure of the grs1 gene is not particularly limited as long as it is a structure that eliminates the function of GRS1, but it preferably contains the following base sequence (a) and / or (b): (a) a nucleotide sequence within an exon constituting a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, which omits all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain of the encoded protein; (b) a nucleotide sequence having 70% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 3;

[0032] The grs1 gene preferably contains, within an exon constituting a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to said amino acid sequence, a nucleotide sequence resulting in a deletion of all or a part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein, and / or a nucleotide sequence showing 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2 or 3.

[0033] The nucleotide sequences shown in SEQ ID NOs: 2 and 3 are examples of the nucleotide sequence of the grs1 gene. Specifically, both of the nucleotide sequences shown in SEQ ID NOs: 2 and 3 have a structure in which a retrotransposon is inserted into the exon sequence of the GRS1 gene. Figure 2 shows the structure of the wild-type GRS1 gene and the retrotransposon insertion positions in the nucleotide sequences shown in SEQ ID NOs: 2 and 3. Figure 2(A) shows the structure of the wild-type GRS1 gene. The GRS1 gene has three exons (exon 1, exon 2, and exon 3) and two introns (intron 1 and intron 2). Figure 2(B) shows the structure of the nucleotide sequence shown in SEQ ID NO: 2. In this sequence, a retrotransposon of approximately 9 kbp has been inserted into the first exon of the GRS1 gene. Figure 2(C) shows the structure of the nucleotide sequence shown in SEQ ID NO: 3. In this sequence, a retrotransposon of approximately 1.2 kbp has been inserted into the third exon of the GRS1 gene.

[0034] The plants of the present invention contain a functionally defective glucoraphasatin synthase gene, which allows them to contain high concentrations of glucoraphanin. It is presumed that in plants of the genus Radish (particularly Japanese radish) that contain a homozygous functionally defective glucoraphasatin synthase gene, the conversion of glucoerucin to glucoraphasatin is inhibited, resulting in the accumulation of glucoerucin. Radish leaves have not been shown to efficiently convert glucoerucin to glucoraphanin, but plants of the genus Brassica (particularly Brassica oleracea) have the ability to biosynthesize glucoraphanin from glucoerucin. The intergeneric hybrid plants of the present invention retain the ability to convert glucoerucin to glucoraphanin inherent in Brassica plants, while suppressing the conversion of glucoerucin to glucoraphasatin, thereby promoting the conversion of glucoerucin to glucoraphanin and allowing them to contain high concentrations of glucophanin.

[0035] The plant of the present invention may be a diploid, but may also be, for example, a triploid or tetraploid allopolyploid. By creating such an allopolyploid, fertility can be restored or improved. Such an allopolyploid may be produced by any known method, for example, by colchicine treatment (see, for example, Zhang et al., Breeding Research, Vol. 3, pp. 31-41 (2001); Ogasawara et al., Sono Gakuken, Vol. 11, No. 2, pp. 189-194 (2012)).

[0036] The plant of the present invention can be used as a food or feed ingredient. As a food, it can be used as a normal vegetable, or it can be made into, for example, a liquid (drink), powder, or granule. Alternatively, it can be used as a material for extracting and purifying glucoraphanin. Purified glucoraphanin can be used, for example, in nutritional supplements, pharmaceuticals, etc.

[0037] 2. Method for producing intergeneric hybrid plants of the Brassicaceae family The method for producing an intergeneric hybrid plant of the Brassicaceae family of the present invention (hereinafter also referred to as the "production method of the present invention") comprises the steps of crossing a first parent plant with a second parent plant and obtaining an intergeneric hybrid plant between the first parent plant and the second parent plant, wherein the first parent plant and the second parent plant are Brassicaceae plants but of different genera, the first parent plant contains glucoraphanin at 5 mg / 100 g FW or more, and the second parent plant contains a functionally defective glucoraphasatin synthase gene.

[0038] In the production method of the present invention, the "first parent plant" is a Brassicaceae plant of a different genus from the second parent plant, containing glucoraphanin at a concentration of 5 mg / 100 gFW or more, preferably 10 mg / 100 gFW or more. While there are no particular limitations on the Brassicaceae plant, as long as it is a Brassicaceae plant of a different genus from the second parent plant, Brassica plants are preferred, and particularly plants containing relatively high amounts of glucoraphanin, such as kale, broccoli, cabbage, kohlrabi, and cauliflower, are preferred. Brassica oleracea is preferred. Such glucoraphanin-rich Brassica oleracea may be obtained, for example, by the method described in Patent Document 1.

[0039] In the production method of the present invention, the "second parent plant" is a Brassicaceae plant that contains a functionally defective glucoraphasatin synthase gene and is classified into a different genus from the first parent plant. There are no particular limitations on the Brassicaceae plant, as long as it is classified into a different genus from the first parent plant, but a plant of the genus Radish is preferred. Raphanus sativus is preferred.

[0040] In the second parent plant, the functionally defective glucoraphasatin synthase (grs1) gene may be contained as a homozygote or a heterozygote. A second parent plant containing the grs1 gene can be selected, for example, by the method described in Patent Document 4. Specifically, a PCR method can be used in which DNA extracted from a sample plant is used as a template, and a primer set that specifically amplifies the GRS1 gene and a primer set that specifically amplifies the grs1 gene are used.

[0041] The second parent plant containing the grs1 gene may be a mutant selected by the above-mentioned method from a large number of progeny lines produced by outcrossing, or a wild-type GRS1 gene modified to eliminate or reduce its function may be used. Any known method can be used to modify the GRS1 gene. For example, mutations can be introduced by introducing an insertion sequence via a transposon, retrotransposon, plant virus, or the like. Other examples of mutation treatments include irradiation of seeds, heavy ion beam treatment, and treatment with a solution containing a mutagen.

[0042] The structure of the grs1 gene contained in the second parent plant is not particularly limited as long as it is a structure that eliminates the function of GRS1, but it is preferable that it contains the following base sequence (a) and / or (b): (a) a nucleotide sequence within an exon constituting a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, which omits all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain of the encoded protein; (b) a nucleotide sequence having 70% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 3;

[0043] The grs1 gene preferably contains, within an exon constituting a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to said amino acid sequence, a nucleotide sequence resulting in a deletion of all or a part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein, and / or a nucleotide sequence showing 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2 or 3.

[0044] When a plant carrying a heterozygous grs1 gene is used as the second parent plant, half of the intergeneric hybrid plants will be functionally defective. In this case, the production method of the present invention preferably includes a step of selecting functionally defective plants. A method for selecting functionally defective plants can be, for example, the method described in Patent Document 4. Specifically, a PCR method can be used in which DNA extracted from a sample plant is used as a template, and a primer set that specifically amplifies the GRS1 gene and a primer set that specifically amplifies the grs1 gene are used.

[0045] In the production method of the present invention, the first parent plant and the second parent plant are classified into different genera, and an intergeneric hybrid plant is obtained by crossing. The crossing method is not particularly limited, and any crossing technique commonly used in breeding and other fields can be used. Furthermore, the plant obtained by crossing may be further subjected to self-crossing for several generations, backcrossed for several generations, or the self-crossing and backcrossing may be repeated as appropriate.

[0046] The production method of the present invention may be a method for producing a diploid, but may also be a method for producing a triploid or tetraploid allopolyploid.

[0047] Unless otherwise specified in this section, the detailed conditions of the parent plants used in the production method of the present invention and the detailed characteristics of the produced plants are the same as those described in section "1. Intergeneric hybrid plants of plants of the genus Brassica and plants of the genus Radish."

[0048] 3. Methods for Producing Cruciferous Plants The method for producing a Brassicaceae plant of the present invention (hereinafter also referred to as the "production method of the present invention") is characterized by comprising the step of cultivating the plant of the present invention described in Section "1. Intergeneric hybrid plant between a plant of the genus Brassica and a plant of the genus Radish." The production method of the present invention makes it possible to obtain a plant with a high glucoraphanin content.

[0049] The plant produced by the production method of the present invention is an intergeneric hybrid plant between a Brassica plant and a Radish plant. Preferably, the plant produced by the production method of the present invention is an edible or forage plant, more preferably an edible vegetable. Preferably, the plant produced by the production method of the present invention is a hybrid between Brassica oleracea and Raphanus sativus, i.e., Raphanobrassica. According to the above preferred embodiment, it is possible to obtain a plant that allows humans or animals (e.g., mammals such as dogs, cats, cows, horses, pigs, sheep, monkeys, and ferrets, and birds such as chickens) to ingest large amounts of Raphanobrassica.

[0050] The plants cultivated by the production method of the present invention may be intergeneric hybrid plants of the F1 generation obtained by crossing a plant of the genus Brassica with a plant of the genus Radish, F2 generation obtained by self-pollinating the F1 generation, or intergeneric hybrid plants obtained by repeated self-pollination from the F2 generation onwards, or intergeneric hybrid plants obtained by backcrossing from the F2 generation onwards.

[0051] 4.Food The food of the present invention is characterized in that it uses as an ingredient the plant of the present invention described in the section "1. Intergeneric hybrid plant of a plant of the genus Brassica and a plant of the genus Radish." As used herein, "food" refers to a substance or composition in a form suitable for human ingestion. The food of the present invention may be the plant itself as a vegetable, or a dish using the vegetable. It may also be, for example, a liquid (drink), powder, granules, etc., processed from the plant.

[0052] 5. Method for increasing the glucoraphanin content of Brassicaceae plants The method of the present invention for increasing the glucoraphanin content of Brassicaceae plants (hereinafter also referred to as the ``high-content method of the present invention'') is characterized by comprising: a first step of preparing a Brassicaceae plant containing 5 mg / 100 g FW or more of glucoraphanin as a first parent plant; a second step of preparing a Brassicaceae plant of a different genus from the first parent plant as a second parent plant, which has a lack of or reduced function of glucoraphanin synthase; and a third step of crossing the first parent plant with the second parent plant.

[0053] In the method for increasing glucoraphanin content of the present invention, the first step is to prepare a Brassica plant as the first parent plant, containing at least 5 mg / 100 g FW of glucoraphanin, preferably at least 10 mg / 100 g FW. The "first parent plant" is not particularly limited as long as it is a Brassica plant classified in a different genus from the second parent plant, but is preferably a Brassica plant, particularly one that contains relatively high amounts of glucoraphanin, such as kale, broccoli, cabbage, kohlrabi, or cauliflower. Brassica oleracea is preferred.

[0054] The method for producing a plant containing 5 mg / 100 g FW or more of glucoraphanin is not particularly limited, and may be, for example, the method described in Patent Document 1. Alternatively, for example, the following method may be used: A large number of progeny lines are produced by outcrossing a Brassica plant. Of the resulting progeny lines, one or more lines containing high concentrations of glucoraphanin are selected, and self-crossing or outcrossing is carried out. Crossing is repeated as necessary to obtain a Brassica plant line with a high glucoraphanin content.

[0055] In the method for increasing glucorafatin content of the present invention, the second step is to prepare a second parent plant, a Brassicaceae plant of a different genus from the first parent plant, in which glucorafatin synthase function is deleted or reduced. The "second parent plant" is not particularly limited as long as it is a Brassicaceae plant of a different genus from the first parent plant, but is preferably a plant of the genus Raphanus. Preferably, it is Raphanus sativus.

[0056] The second parent plant is a plant lacking or reducing the function of glucoraphasatin synthase (GRS1). More specifically, it is a plant containing a functionally defective glucoraphasatin synthase (grs1) gene. The grs1 gene may be contained as a homozygote or heterozygote. A second parent plant containing the grs1 gene can be selected, for example, by the method described in Patent Document 4. Specifically, a PCR method can be used in which DNA extracted from a sample plant is used as a template, and a primer set that specifically amplifies the GRS1 gene and a primer set that specifically amplifies the grs1 gene are used.

[0057] The second parent plant containing the grs1 gene may be a mutant selected by the above-mentioned method from a large number of progeny lines produced by outcrossing, or a wild-type GRS1 gene modified to eliminate or reduce its function may be used. Any known method can be used to modify the GRS1 gene. For example, mutations can be introduced by introducing an insertion sequence via a transposon, retrotransposon, plant virus, or the like. Other examples of mutation treatments include irradiation of seeds, heavy ion beam treatment, and treatment with a solution containing a mutagen.

[0058] The third step of the method for increasing the content of a plant of the present invention is to cross the first parent plant with the second parent plant. The crossing method is not particularly limited, and any crossing technique commonly used in breeding and the like can be used. Furthermore, the plant obtained by crossing may be further subjected to self-crossing for several generations, backcrossing for several generations, or repeated self-crossing and backcrossing as appropriate.

[0059] The method of the present invention for increasing glucoraphanin content by the above steps can increase the glucoraphanin content of a Brassicaceae plant. The Brassicaceae plant obtained by the method of the present invention for increasing glucoraphanin content preferably contains at least 20 mg / 100 g FW, at least 30 mg / 100 g FW, at least 50 mg / 100 g FW, at least 100 mg / 100 g FW, or at least 150 mg / 100 g FW.

[0060] Unless otherwise specified in this section, and unless otherwise contradicted, the detailed conditions of the parent plants, etc. used in the high-content method of the present invention, and the detailed characteristics of the plants produced are the same as the conditions, characteristics, etc. described in sections "1. Intergeneric hybrid plants of Brassica plants and Radish plants" and "2. Method for producing Brassicaceae plants." [Example]

[0061] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0062] [Example 1] Creation of intergeneric hybrid plants (Raphanobrassica) Intergeneric hybrid plants between plants of the genus Raphanus and plants of the family Brassicaceae were produced using the following procedure. The kale line "KK-45," which contains glucoraphanin, was used as the Brassicaceae plant. The commercially available radish cultivar "Nishimachi Riso" was used as the plant of the genus Raphanus. Nishimachi Riso is known to contain a mixture of individuals heterozygous for both functional and non-functional GRS1 genes within the cultivar (see Patent Document 4). DNA marker testing of Nishimachi Riso was performed in advance, and heterozygous individuals (hereinafter also referred to as "AKO") were selected at the seedling stage. Four strains (AKO103, AKO108, AKO110, and AKO118) were used as seed parents for hybridization. The cultivars and lines used and the intergeneric hybrids produced are shown in Table 1.

[0063] [Table 1]

[0064] DNA marker testing of Nishimachi Riso and each hybrid progeny was performed as follows: DNA was extracted from Nishimachi Riso leaves, and PCR reactions were performed using a primer set consisting of the three primers shown in Table 2. Observation of 392-bp DNA amplification indicated the presence of a "deficient" gene, and observation of 222-bp DNA amplification indicated the presence of a "functional" gene.

[0065] [Table 2]

[0066] In Nishimachi Ideal, individuals showing both 392bp and 222bp DNA amplification were determined to be "heterozygous," while individuals showing only 222bp DNA amplification were determined to be "wild-type." DNA marker testing for each cross progeny was performed using the following procedure. Among the cross progeny, individuals carrying the functional GRS1 gene were determined to be "functional" cross progeny, and individuals carrying the loss-of-function GRS1 (grs1) gene were determined to be "loss-of-function" cross progeny.

[0067] [Example 2] Measurement of various glucosinolate contents in leaves Samples used for glucosinolate content analysis were prepared as follows. Three 20 cm long true leaves were collected from each plant in the field. The tip 10 cm of each leaf blade was removed, and the vein running down the center of the leaf was removed. The samples were freeze-dried for 4–5 days (Labconco freeze dryer), and the dried samples were crushed (Yasui Kikai Multi-Beads Shocker). 0.1 g of dried powder was weighed out, 5 mL of 80% methanol was added, and the mixture was shaken and stirred at room temperature for 30 minutes. The supernatant obtained after centrifugation at 3000 rpm for 10 minutes was used as the glucosinolate extract. The glucosinolate extract was adsorbed onto a DEAE Sepharose column and desulfurized with acid sulfatase (25°C for 18 hours). The desulfurized glucosinolates were eluted with ion-exchanged water to obtain a desulfo-glucosinolate solution. The desulfo-glucosinolate solution was subjected to HPLC under the following conditions, and a chromatogram was obtained at a UV detection wavelength of 229 nm. ·Equipment used: LC-20A, Shimadzu Corp., Japan Column type: COSMOSIL 5C18-II, 150 x 4.6 mm, Nacalai-Tesque Inc., Japan Mobile phase solvent composition: 20% acetonitrile Sample injection volume: 20 μl ·Flow rate: 1.5 mL / min Column temperature: 30℃ The contents of each glucosinolate (glucoraphanin, glucoraphenin, glucoerucin, glucoraphasatin) in the sample were calculated based on the results of HPLC analysis of each glucosinolate standard sample with known concentrations under the same conditions. The measurement results of each glucosinolate for each hybrid combination are shown in Table 3.

[0068] [Table 3]

[0069] Within all cross combinations, the average glucoraphanin content of the populations carrying the loss-of-function type was higher than that of the populations carrying the functional type. The ratio of glucoraphanin content of the loss-of-function type to that of the functional type ranged from 1.97 to 2.56. Loss-of-function individuals contained very little or no glucoraphenin and glucoraphasatin. These results demonstrate that suppressing GRS1 expression in Raphanobrassica results in the metabolism of much of the glucoerucin into glucoraphanin, increasing the glucoraphanin content.

[0070] [Example 3] Measurement of glucosinolate content in roots, buds, and stems Root, bud, and stem samples for analysis of glucosinolate content were prepared using the following procedure. Ten individual plants of each species were dug up from the roots in the field and washed with water. Roots were cut into 0.5-1 cm thick slices approximately 5 cm below the junction between the stem and hypocotyl. Terminal flower buds were collected from 10 bolted individual plants of each species. Stems were collected approximately 10 cm below the junction with the bud from 10 bolted individual plants of each species. Each sample was freeze-dried for 4-5 days, and the dried samples were crushed. Extraction, desulfurization, and HPLC analysis were then performed using the same procedures as in Example 2. The measurement results for each glucosinolate in each sample are shown in Table 4.

[0071] [Table 4]

[0072] As shown in Table 4, the average glucoraphanin content was higher in the functional individuals than in the functional individuals in all parts. Furthermore, the functional individuals contained very little or no glucoraphenin and glucoraphasatin. These results demonstrate that suppressing the expression of the GRS1 gene in Raphanobrassica increases the glucoraphanin content, regardless of the harvesting site.

[0073] [Example 4] Correlation of glucoraphanin content in seeds and leaves of cruciferous vegetables Seed and leaf samples of cruciferous vegetables were prepared for analysis of glucoraphanin content using the following procedure. A total of 68 lines of cruciferous vegetables, including KK-45 and commercially available kale varieties, were used. Three true leaves measuring 20 cm in length were harvested from each individual plant in a greenhouse. The tip of the leaf blade (10 cm) was harvested from each leaf, and the vein running through the center of the leaf was removed. Seeds were harvested from the same plants from which the leaves were harvested, and 0.5 g of seeds were used per plant. Each sample was freeze-dried for 4 to 5 days, and the dried sample was crushed. Extraction, desulfurization, and HPLC analysis were then performed using the same procedures as in Example 2. The HPLC analysis conditions were as follows: ·Equipment used: LC-20A, Shimadzu Corp., Japan Column type: COSMOSIL 5C18-II, 150 x 4.6 mm, Nacalai-Tesque Inc., Japan Mobile phase solvent composition: 20% acetonitrile Sample injection volume: 20 μl ·Flow rate: 1.5 mL / min Column temperature: 30℃ The correlation between the HPLC peak area (content) of glucoraphanin in samples derived from leaves and seeds of each individual plant is shown in Figure 3 .

[0074] As shown in Figure 3, a correlation was observed between the HPLC peak areas of glucoraphanin in samples derived from the leaves and seeds of Brassicaceae vegetables, indicating that varieties with high glucoraphanin content in their leaves also have high glucoraphanin content in their seeds. These results suggest that even in intergeneric hybrid plants of the Brassicaceae family, individuals with high glucoraphanin content in their leaves also have high glucosinolate content in their seeds. [Industrial Applicability]

[0075] The present invention is applicable to agriculture, food manufacturing, pharmaceutical manufacturing, and the like. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An intergeneric hybrid plant of a Brassica plant and a Radish plant, The Brassica plant contains glucoraphanin in an amount of 5 mg / 100 g (fresh weight) or more, The plant of the genus Radish contains a functionally defective glucoraphasatin synthase gene, The intergeneric hybrid plant has a ratio of glucoraphanin content to glucoraphenin content of 1.0 or more, The glucoraphanin content is 20 mg / 100 g (fresh weight) or more, A plant containing a functionally defective glucoraphasatin synthase gene.

2. 2. The plant according to claim 1, wherein the glucoraphenin content is 50 mg / 100 g (fresh weight) or less.

3. The plant according to claim 1 or 2, wherein the function-deficient glucoraphasatin synthesis gene is the following gene (a) and / or (b): (a) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, wherein the gene is accompanied by a deletion of all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein within an exon constituting the gene, and the deletion results in a loss of the glucoraphasatin synthesis function of glucoraphasatin synthase; (b) A gene containing a base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 2 or 3.

4. The plant according to any one of claims 1 to 3, wherein the Brassica plant is Brassica oleracea.

5. The plant according to any one of claims 1 to 4, wherein the plant of the genus Raphanus is Raphanus sativus.

6. The plant according to any one of claims 1 to 5, having polyploidized chromosomes.

7. 1. A method for producing an intergeneric hybrid plant of the Brassicaceae family, comprising: crossing a first parent plant with a second parent plant; obtaining an intergeneric hybrid plant between the first parent plant and the second parent plant. the first parent plant is a Brassica plant containing glucoraphanin at a concentration of 5 mg / 100 g (fresh weight) or more; the second parent plant is a plant of the genus Radish containing a functionally defective glucoraphasatin synthase gene, The method, wherein the ratio of the glucoraphanin content to the glucoraphenin content of the intergeneric hybrid plant is 1.0 or more, and the glucoraphanin content is 20 mg / 100 g (fresh weight) or more.

8. The method according to claim 7, wherein the functionally defective glucoraphasatin synthase gene is the following gene (a) and / or (b): (a) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence showing 90% or more sequence identity to said amino acid sequence, wherein the gene is accompanied by a deletion of all or part of the 2-oxoglutarate-iron(II)-dependent oxygenase domain in the encoded protein within an exon constituting the gene, and the deletion results in a loss of the glucoraphasatin synthesis function of glucoraphasatin synthase; (b) A gene containing a base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 2 or 3.

9. The method according to claim 7 or 8, further comprising a step of selecting an intergeneric hybrid plant containing a functionally defective glucoraphasatin synthase gene from the intergeneric hybrid plants.

10. The method according to any one of claims 7 to 9, wherein the first parent plant is a Brassica plant and the second parent plant is a Radish plant.

11. 11. The method of claim 10, wherein the Brassica plant is Brassica oleracea.

12. The method according to claim 10 or 11, wherein the Raphanus plant is Raphanus sativus.

13. The method according to any one of claims 7 to 12, wherein the intergeneric hybrid plant comprises polyploidized chromosomes.

14. A method for producing a Brassicaceae plant, comprising the step of cultivating the intergeneric hybrid plant according to any one of claims 1 to 6.

15. A food product made from the intergeneric hybrid plant according to any one of claims 1 to 6 as a raw material.

16. A method for increasing the glucoraphanin content of a Brassicaceae plant, comprising: A first step of preparing a Brassica plant containing glucoraphanin at a concentration of 5 mg / 100 g (fresh weight) or more as a first parent plant; A second step of preparing a plant of the genus Radish lacking the function of glucoraphasatin synthase as a second parent plant; and a third step of crossing the first parent plant with the second parent plant; A method in which the ratio of the glucoraphanin content to the glucoraphenin content of the obtained Brassicaceae plant is 1.0 or more, and the glucoraphanin content is 20 mg / 100 g (fresh weight) or more.

17. The method according to claim 16, wherein the second step comprises modifying the glucoraphasatin synthase gene to render the glucoraphasatin synthase function defective.

Images