JP2007060992A - Method for producing anthocyanin-rich root vegetable - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing root vegetables with rich anthocyanin in the hypocotyls or roots thereof (e.g. red radish, red turnip), and seeds thereof. <P>SOLUTION: The method for producing anthocyanin-rich root vegetables comprises the following steps (1) to (5): The step (1): the seeds of root vegetables containing anthocyanin in the hypocotyls or roots thereof or the seeds collected in a step (4) described below are sawn and sprouted and raised. The step (2): part of the hypocotyls or roots of the root vegetables which are obtained in the step (1) are cut off and the amount of anthocyanin in the segments is measured. The step (3): the root vegetables derived from anthocyanin-rich segments are selected, raised again and grown to the flowering stage. The step (4): the resulting flowered root vegetables are crossed with different individuals to collect seeds. The step (5): the resultant seeds in the step (4) are collected or supplied to the step (1). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing root vegetables having a high anthocyanin content. Specifically, the present invention relates to a method for producing red radish and red turnip with a large amount of anthocyanins contained in the hypocotyl and root.

  Anthocyanins are red natural pigments widely used for coloring foods and the like. Recently, however, not only the coloring function but also antioxidant, antimutagenicity, blood pressure lowering, visual improvement, liver dysfunction reduction, antidiabetic, etc. It has come to be noticed as.

    Anthocyanins are widely distributed in higher plants than fern plants such as perilla, sweet potato (purple potato), red cabbage, red turnip, colored potato, red radish, grape and berry. In general, anthocyanins exhibit a bright red color in the acidic region, but gradually become bluish as pH increases from weakly acidic to neutral to weakly alkaline. However, since anthocyanins contained in red radish can maintain a clear red color even in neutral to weakly acidic regions (Patent Document 1), anthocyanins derived from these plants are red pigments (red radish pigments). ), And its efficient production is required.

    As a method for efficiently obtaining a natural pigment, a method of producing a plant containing the pigment in a high ratio can be considered.

    Conventionally, as a method for obtaining a plant having a high pigment content, a method for obtaining a chlorella mutant having a high chlorophyll and a high carotenoid content is known. Specifically, the parent strain chlorella was mutated by gently shaking at 30 ° C. for 60 minutes in the presence of 50 μg / ml nitrosoguanidine, and green color was visually detected from the obtained mutant strains. Is selected (Patent Document 2).

However, there is no known method for obtaining plants having a high anthocyanin content, particularly root vegetables.
JP 09-154532 A JP-A-11-75823

  An object of the present invention is to provide a root vegetable having a high anthocyanin content, such as red radish and red turnip, and a method for producing the seed. Furthermore, this invention aims at providing the root vegetables produced by such a method, and its seed.

  The anthocyanin content of red radish hypocotyls and roots (hereinafter also referred to simply as “roots”) belonging to root vegetables is different between the surface and the interior, and the content of the root surface is high, but the content of the interior is low. Individuals are also frequently present. Nevertheless, conventional selection of red radish with a high content of anthocyanins is usually carried out by visual observation in the field, focusing only on the red shade of the root surface. In view of this situation, the present inventors should develop a method for more accurately selecting red radish with a high anthocyanin content, using the internal anthocyanin content as a direct index rather than the shade of the surface color of red radish roots. During my research, I found that growth, mating and seeding can be carried out normally even when red radish lacking a part of the root is used. Based on this finding, the anthocyanin content of the red radish slices cut from the roots was actually measured to select the radish having a high anthocyanin content, and the selected red radish was crossed to increase the anthocyanin in the root. It was confirmed that red radish can be produced including the ratio.

The present invention has been completed based on such findings, and includes the following aspects:
Item 1. A method for producing seeds of root vegetables with a high content of anthocyanins, comprising performing the following steps (1) to (5):
(1) a step of sowing, germinating and growing seeds of root vegetables containing anthocyanins in the hypocotyl or root, or seeds collected in the following step (4);
(2) A step of cutting a part of the hypocotyl or root part of the root vegetable obtained in the step (1) and measuring the amount of anthocyanin contained in the slice,
(3) selecting a root vegetable derived from a section having a high anthocyanin content, growing it again, and growing it until the flowering stage;
(4) The flowering root vegetable obtained in step (3) is crossed or pollinated between different individuals, and the seed is collected, and (5) the seed obtained in step (4) is collected, or the above The process used for a process (1).

Item 2. Item 2. The method according to Item 1, wherein the root vegetable selected in the step (3) contains anthocyanin at a ratio of 6 mg / cm ³ or more in 1 cm ³ of the hypocotyl or root section.
Item 3. Item 3. The method according to Item 1 or 2, wherein the root vegetable is red radish or red turnip.
Item 4. Item 4. A root vegetable seed having a high anthocyanin content produced by the method according to any one of Items 1 to 3.

Item 5. Item 5. A method for producing a root vegetable having a high content of anthocyanins, comprising a step of seeding and germinating seeds according to Item 4.
Item 6. Item 6. A root vegetable with a high content of anthocyanins produced by the method according to Item 5.

  According to the present invention, a root vegetable (eg, red radish or red turnip) having a high anthocyanin content, particularly a high anthocyanin content per unit volume, and its seeds are compared with the conventional selective mating method by surface color tone. Can be acquired well and efficiently.

The present invention will be described below.
I. TECHNICAL FIELD The present invention provides a method for producing a root vegetable seed that contains anthocyanin in the hypocotyl or root, and contains a high percentage of anthocyanin.

  Here, examples of root vegetables containing anthocyanins in the hypocotyl or root include red radish and red turnip.

  Red radish is a variety of radish belonging to the genus Raphanus sativus L .. The red radish targeted by the present invention contains anthocyanins in the hypocotyl and / or root, and is not limited as long as it is specifically exemplified, but specific examples include red radish, Shin Satomi, Iwakuni Red, etc. be able to.

  Red turnip is a cultivar of turnip belonging to Brassica Rapa L .. The red turnip targeted by the present invention contains anthocyanins in the hypocotyl or / and root, and is not limited as long as it is specifically exemplified. can do.

  The method of the present invention can be carried out by performing the following steps (1) to (5).

a. Process (1)
In the step (1), seeds of root vegetables containing anthocyanins are sown on the hypocotyl or root, germinated and grown.

  Here, the root vegetable seeds may be root vegetable seeds containing anthocyanins in the hypocotyl or root, such as the above red radish and red turnip. Specifically, the various radish and red turnip seeds described above can be preferably used.

  Seeds are usually sown in soil. Cultivation up to sowing, germination and growth can be carried out in the same manner as normal root vegetable cultivation. Although it is not limited, specifically, after sown seeds in the middle of September, the cultivation method of cultivating as a two-row planting of about 20 cm between strains, about 140 cm cocoon width, about 40 cm inter-row spacing may be exemplified. it can.

The growth may be carried out until the hypocotyl or root portion of the root vegetable reaches a volume of at least 20 cm ³ , but it is usually carried out until the hypocotyl or root portion protruding from the soil no longer enlarges.

  Usually, about 70 to 100 days from sowing can be mentioned as a period required to grow until the hypocotyl or the root protruding from the soil does not further enlarge. Specifically, as described above, for example, when seeds are sown in the middle of September, the seeds can be grown from the beginning of December to the end of December.

  Next, the growth is temporarily interrupted at this stage, and the following step (2) is performed. In addition, operation of the said process (1) can be performed similarly about the seed extract | collected at the process (4) mentioned later, and is similarly provided to the following process (2).

b. Process (2)
In the step (2), a part of the hypocotyl or root part of the root vegetable obtained in the above step (1) is cut out, and the amount of anthocyanin contained in the slice is measured.

  There are no particular limitations on the method and amount of cutting off the hypocotyl or root part of the root vegetable, unless the hypocotyl or root excision hinders subsequent growth, flowering, mating (pollination) and fruit set. In order to more accurately measure the amount of anthocyanin contained in the roots of root vegetables, it is preferable to cut so that the hypocotyl or the central part of the root part to the surface is uniformly contained. Taking red radish as an example of root vegetables, as shown in FIG. 1, as a preferable cutting method, when the root is considered as a cone, the top cross-sectional circle is divided into a plurality of lines intersecting the center. A method may be mentioned in which the divided section is cut from the top to the bottom as it is, and a section (FIG. 2) having a substantially triangular cubic shape is cut out.

  The weight and size of the section to be cut are not particularly limited, but it is desirable to cut at least 5 g, preferably 7 to 10 g. To this extent, the volume of the section to be cut out is not particularly limited. For example, in the case of a root vegetable grown until the hypocotyl or the root projecting from the soil does not grow any more, 5 to 5 of the total volume of the hypocotyl and the root combined. Cut out sections of volume corresponding to 10% by volume, preferably 6-9% by volume. Usually, when a section is cut out by the above-described cutting method, a maximum of 16 divisions can be given as the number of divisions of a circle in the cross section. That is, at least 1/16 of the total volume including the hypocotyl and root is cut out so as to uniformly include from the center to the periphery. In addition, when dividing as described above, the limit of the size to be cut out is about 8 divisions, that is, about 1/8 of the total volume including the hypocotyl and root, and 1/8 divided as described above is cut out. However, it has been confirmed that it does not affect the subsequent mating and fruiting.

  The measurement of the amount of anthocyanin contained in the section is not particularly limited, but usually, anthocyanin is extracted from the cut section, and the amount of anthocyanin contained in the obtained anthocyanin extract is measured using a conventional means such as HPLC. A method can be mentioned. Extraction of anthocyanins from root vegetable slices is not limited, but, for example, the cut slices are further finely cut and immersed in a 4-fold weight 0.5% sulfuric acid aqueous solution and left in a dark place for 12 hours. Can be mentioned. The method for quantifying anthocyanins by HPLC can be specifically exemplified by the methods given in the examples, but is not limited thereto as long as it is a method that can quantitate anthocyanins.

c. Step (3)
In step (3), a root vegetable derived from a section having a high anthocyanin content is selected from the measurement result of the anthocyanin content in step (2), and this is grown again and grown until the flowering stage.

Here, the section having a high anthocyanin content basically means a section having a higher anthocyanin content than others in the screening target group in a relative evaluation among a plurality of root vegetable sections to be screened. Preferably, in an absolute evaluation, an anthocyanin amount contained in 1 cm ³ of a slice can be 6 mg / cm ³ or more. A preferable amount of anthocyanin is 7 mg / cm ³ or more per 1 cm ³ of the slice, more preferably 9 mg / cm ³ or more.

  Under such an index, a root vegetable derived from a section having a high anthocyanin content is selected, planted again in the soil, and continued to grow until the flowering stage. In addition, the conditions demonstrated by process (1) can be similarly used for the growth conditions at this time.

d. Step (4)
In the step (4), the flowering root vegetables obtained in the above step (3) are crossed (pollinated) between different individuals, and seeds are collected after fruiting. In addition, it is preferable to perform crossing (pollination) among as many individuals as possible. Crossing (pollination) can be performed artificially, but can also be performed through insects. In the case of an insect medium, in order to cross (pollinate) among as many individuals as possible, there can be mentioned a method in which a net is placed over a plurality of individuals to be crossed (pollinated) and honey bees are released therein.

e. Process (5)
Step (5) is a step of collecting the seeds obtained in the above step (4). The collected seeds can be used as root vegetables seeds with high anthocyanin content, commercially, or for root vegetables cultivation.

  Moreover, the said seed can also be used for the said process (1) again. By subjecting it to the above step (1) again and repeating (1) to (4) once or more, preferably 2 times or more, more preferably 3 to 4 times, finally, a more preferable anthocyanin-rich root vegetable Seeds can be selected and produced. In addition, when the steps (1) to (4) are repeated a plurality of times, the determination of whether the slice has a high anthocyanin content is performed by using the amount of anthocyanin in the parent individual before mating as an index and comparing it with the content. .

  Thus, the seeds obtained by the method of the present invention can be effectively used for the cultivation of these root vegetables as seeds of root vegetables with a high content of anthocyanins, preferably red radish with a high content of anthocyanins, or seeds of red turnips. .

  Cultivation and acquisition of root vegetables with a high content of anthocyanins can be performed by sowing seeds produced by the above-described method of the present invention according to a conventional method and germinating them.

The root vegetables thus obtained, particularly red radish or red turnip, contain a high content of anthocyanins in their hypocotyls or roots. The ratio of the high content is not particularly limited, but preferably the anthocyanin content in the hypocotyl or root is 6 mg / cm ³ or more, preferably 7 mg / cm ³ or more, more preferably 9 mg / cm ³ or more. .

  Such root vegetables are useful as foods (vegetables) themselves, and can be effectively used as a raw material for collecting pigments and anthocyanins, which are bioregulatory functional food materials, and as a processing raw material for bioregulatory functional foods.

  Examples, comparative examples, and experimental examples are described below in order to clarify the configuration and effects of the present invention. However, the present invention is not limited to these examples.

Example 1
1. Selection As seeds to be used in the following process, red radish seeds (hereinafter referred to as “unselected population”) introduced into Japan from Yunnan, China, and the color and size of the epidermis from the unselected population Based on the above, seeds seeded after three selections in the field (hereinafter referred to as “three-time selection population”) were used.

(1) Process 1
The seeds (non-selected group, three-time selected group) were sown in a field in Osaka University Medicinal Botanical Garden in mid-September. The plant was cultivated as a two-row planting of 20 cm between strains, 140 cm in width, and 40 cm in width. After confirming that the hypocotyls or roots protruding from the soil were no longer enlarged, the plants were harvested in mid-December.

(2) Process 2
As shown in FIG. 1, a section (FIG. 2) corresponding to about 1/16 of the individual volume was cut out from the hypocotyl and root of the harvested red radish so as to include from the surface to the center. In addition, the individual | organism | solid which cut out the hypocotyl and a part (slice) of a root part was returned to the field within 1 hour after cutting, and it transplanted to soil again.

  The sections were cut into approximately 5 mm squares, immersed in a 0.5% aqueous sulfuric acid solution 4 times the cut weight, and allowed to stand in the dark for 12 hours. After standing, the immersion liquid was collected by filtration, and the amount of anthocyanin contained in the anthocyanin extract was analyzed and measured by HPLC. HPLC analysis consists of various units (2 pumps (PU2080), high pressure gradient unit (MX-2080-32), autosampler (AS2057), column oven (CO2060), photodiode array detector (MD2010), 3 channel solvent) Using an HPLC system Borwin (JASCO) composed of a degasser (DG-2080-53) and a chromatogram data recording device (JASCO Bowin software), the following conditions were used.

<HPLC conditions>
Column : Develosil C30 UG-5 (4.6 id x 250 mm, Nomura Chemical Co., Ltd.)
Mobile phase : A: 0.1% TFA-CH ₃ CN, B: Water (Increase the ratio of A from 0% → 25min → 65min to 0% → 20% → 30%)
Flow rate : 1.0 mL / min
Column temperature : 30 ° C
Detection wavelength : 515 nm
Sample injection volume : 20 μL.

  Measurement (quantitative) of the amount of anthocyanins was performed by the absolute calibration curve method based on the peak area of HPLC. From the obtained results, both the anthocyanin content contained per unit volume of red radish and the anthocyanin content contained per red radish individual were calculated.

A standard calibration curve was prepared by subjecting the red radish anthocyanin mixture to HPLC under the above analytical conditions at the ratio of 5, 25, 50, 100, and 200 ng. Here, the red radish anthocyanin mixture was adsorbed on 38 cm ³ of the DIAION HP-20 column after diluting 3 mL of VEGETA RED AD (San-Eigen FFI Co., Ltd.) with 12 mL of 0.1% TFA aqueous solution. After eluting sequentially with 0.1% TFA aqueous solution, 10% MeOH-0.1% TFA aqueous solution, and 100% MeOH-0.1% TFA, the prepared eluate with 100% MeOH-0.1% TFA was concentrated and prepared. used.

(3) Process 3
Based on the above analysis results, the top 2 individuals with the highest anthocyanin content (mg / cm ³ ) per unit volume from each population (unselected population, 3 selected population), and anthocyanin content per individual ( The top 2 individuals with high content of (g / individual) were selected. Individuals selected from the above groups (highly selected individuals / unit volume, high content selected individuals / individuals) from among the individuals that had been re-transplanted to the field (individuals that were cut out of the hypocotyl and root) It was transferred to a pot filled with soil in the field, cultivated in a vinyl house, and flowered in the spring of the following year.

For control purposes, 2 individuals (control individuals / unit volume) with an average anthocyanin content (mg / cm ³ ) per unit volume from each population (unselected population, 3 times selected population), and 1 individual Two individuals (control individuals / individuals) whose average anthocyanin content (g / individual) was selected were cultivated in the same manner as described above, and flowered in the spring of the following year.

(4) Process 4
For each group, mating was carried out between the two selected individuals flowered in Step 3 (high-content selected individuals / unit volume, high-content selected individuals / individuals, control individuals / unit volumes, control individuals / individuals). The mating was performed artificially using a fine brush.

(5) Process 5
From each individual mated in the above step 4 (high-content selected individual / unit volume, high-content selected individual / individual, control individual / unit volume, control individual / individual), seeds were collected after ripening. The seeds were harvested together with the berries, and then the berries were crushed by hand.

2. Measurement of anthocyanin content contained in the mating individual The seeds of each mating individual (high content selected individual / unit volume, high content selected individual / individual, control individual / unit volume, control individual / individual) collected in the above step 5 (1) As described in Step 1, seeded in a field in Osaka University Medicinal Botanical Garden, cultivated as a double-row planting of 20 cm between strains, 140 cm wide, 40 cm long, and the hypocotyl or root protruding from the soil It was harvested after confirming that it was no longer enlarged. For the harvested red radish, the anthocyanin content in the hypocotyl and root is measured by HPLC according to the method described in step 2 above (2), and the anthocyanin content per unit volume of red radish, Both anthocyanin contents contained in the perimeter were calculated.

2. Results Table 1 shows the anthocyanin content [content per unit volume (mg / cm ³ ), content per individual (g / individual)] of the crossed individuals obtained by selecting the unselected population. In the table, “mating individual 1” means an individual obtained by mating between individuals containing high anthocyanins per unit volume in the unselected population (highly selected individual / unit volume). “2” means individuals obtained by crossing between individuals with high anthocyanin content (highly selected individuals / individuals) per individual in the unselected population.

  For comparison, selected from individuals obtained by mating between individuals with average anthocyanin content (control individuals / unit volume, control individuals / individuals) and unselected populations (seed) The anthocyanin content per unit volume and per individual is shown together for individuals obtained by cultivating the selected seeds (indicated as “original population” in the table).

Table 2 shows the anthocyanin content (content per unit volume (mg / cm ³ ), content per individual (g / individual)) of the mating individuals obtained by selecting the three selected populations. In the table, “mating individual 3” means an individual obtained by mating between individuals containing high anthocyanins per unit volume (highly selected individual / unit volume) in a population selected three times. “Individual 4” means an individual obtained by crossing between individuals with high anthocyanin content (highly selected individual / individual) per individual in a three-time selection population.

  For comparison, individuals obtained by mating individuals (control individuals / unit volume, control individuals / individuals) having an average anthocyanin content from three selected populations (indicated as “control” in the table), and 3 The anthocyanin content per unit volume and per individual is also shown for individuals obtained by cultivating seeds arbitrarily selected from the twice-selected population (seed) (indicated as “original population” in the table).

  As shown in Tables 1 and 2, the anthocyanin content is the individual obtained by mating between the selected individuals, or the original population in the individual obtained by mating between the selected individuals, both per unit volume and per individual. The value was higher than the average value. In addition, the anthocyanin content of the crossed individuals was higher than that of the control, and it was shown that the high productivity of the anthocyanins of the crossed individuals is maintained in the next generation (Table 1). Furthermore, compared to the original population, the anthocyanin content of the mating individuals is 2.0 times per unit volume and 1.7 times per individual in the unselected population (Table 1), 1.4 times per unit volume and 1.9 per individual in the triple selected population. Doubled (Table 2).

  From these results, the method of the present invention for selecting and mating individuals with high anthocyanin content by cutting roots as the site to be used for anthocyanin extraction, compared with the conventional method of selective mating by surface color, It was revealed that the anthocyanin content was dramatically improved.

It is a figure which shows one method which cuts out the section for anthocyanin content measurement from the hypocotyl and root part of red radish so that it may include from the surface to a center part. Here, as an example, a method of cutting out a section corresponding to about 1/16 of the individual volume is shown. Sections corresponding to about 1/16 of the individual volume cut out above are shown.

Claims (6)

A method for producing seeds of root vegetables with a high content of anthocyanins, comprising performing the following steps (1) to (5):
(1) a step of sowing, germinating and growing seeds of root vegetables containing anthocyanins in the hypocotyl or root, or seeds collected in the following step (4);
(2) A step of cutting a part of the hypocotyl or root part of the root vegetable obtained in the step (1) and measuring the amount of anthocyanin contained in the slice,
(3) selecting a root vegetable derived from a section having a high anthocyanin content, growing it again, and growing it until the flowering stage;
(4) The flowering root vegetable obtained in step (3) is crossed or pollinated between different individuals, and the seed is collected, and (5) the seed obtained in step (4) is collected, or the above The process used for a process (1). The method according to claim 1, wherein the root vegetable selected in step (3) contains anthocyanin at a ratio of 6 mg / cm ³ or more in 1 cm ³ of hypocotyl or root section.   The method according to claim 1 or 2, wherein the root vegetable is red radish or red turnip.   A root vegetable seed having a high anthocyanin content produced by the method according to any one of claims 1 to 3.   A method for producing a root vegetable having a high anthocyanin content, comprising a step of sowing and germinating and growing the seed according to claim 4.   A root vegetable with a high content of anthocyanins produced by the method according to claim 5.

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