JP2957128B2 - Enlargement culture method of wasabi rhizome - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel culture method for enlarging wasabi rhizomes by tissue culture. More specifically, the present invention relates to a wasabi of the brassicaceae such as wasabi and so on, which has conventionally taken a long time to grow. Plants called
Tissue culture using a specific medium and culturing conditions makes it possible to remarkably enlarge the wasabi rhizome in a short period of time and increase the rhizome sinigrin content of the wasabi plant. It is about.

[0002]

2. Description of the Related Art Wasabi is a spice that has been widely used as a seasoning for fish meat and the like since ancient times. Like spicy mustard, the main body of spiciness is glucosinolate (mainly sinigrin) and myrosinase contained in wasabi. Isothiocyanate (mainly allyl isothiocyanate) produced by the enzymatic reaction of Above all, sawa wasabi has a very high value as a spice with excellent pungency and flavor. In addition, horseradish is an important plant used as a raw material for preparing an industrially useful peroxidase, although it has an inferior flavor to the wasabi, but contains the same pungent component as the wasabi. . Conventionally, the method of breeding the wasabi plant includes a method using seedlings and a method using stock division. The former is mainly used as a sexual propagation method (seedling method) in which seedlings are grown from seeds in a seedling growing house or the like. The latter is widely known as an asexual propagation method (straining method) in which the rhizome of the parent strain is divided and bred in wasabi fields.

[0003] By the way, the conventional seedling method is generally less susceptible to soft rot and inking disease, and can produce uniform seedlings. However, it is difficult to breed a large number of excellent genetically uniform strains, and it takes a long time to raise seedlings, which requires a nursery bed for raising seedlings. On the other hand, the stock splitting method uses sprouts that are removed at the time of harvest, so that a seedbed for raising seedlings is unnecessary, and because the trait of the parent strain is inherited as it is, it is suitable for breeding genetically uniform excellent strains. However, it is easy to be infected with soft rot and inking disease because the rhizome is wounded by the division, and the number of seedlings obtained depends on the number of rhizomes. It is difficult. Further, when the parent strain is infected with a viral disease, there is a drawback that the whole divided seedlings have a high risk of being infected with the viral disease.

[0004] In order to solve the problems of the prior art, recently, generally, as a technique for mass propagation of plants,
Various methods have been studied for mass-producing wasabi by using a tissue culture technique that has remarkably advanced. Mass propagation by tissue culture is expected to be a useful method for resolving disadvantages, taking advantage of vegetative propagation, but is generally used as a method for growing seedlings using such tissue culture. Is widely used by the mericlone method (growth point culture). In this Mericlone method, the stem apex of a plant is cultured in a tissue culture medium to obtain Mericlone (a young plant),
This is a method in which the cultivation and division of the mericlone are repeated to obtain a large number of mericlones, and the mericlone is transformed into seedlings in a predetermined medium.

[0005] Techniques for increasing the number of above-ground parts (leaves and petiole parts) of wasabi plants by using the mericlone method and thereby mass-producing seedlings have produced a number of research results. [For example, Hidetaka Hori: Mass culture method of wasabi seedlings in vitro, Hyundai Kagaku, 5 (extra), 118-123 (1986), Takashi Hosoki et al .: Propagation of Wasabi by Tissue Culture, Agriculture and Horticulture, 61
(8), 996-996 (1986), Otsuka Toshio: Wasabi propagation method, agriculture and horticulture, 63 (1), 185-1
89 (1988); Takashi Hosogi et al .: Propagation of Wasabi Tissue Culture Seedling, Agriculture and Horticulture, 63 (5), 653-654.
(1988)].

[0006] As a method for growing wasabi seedlings to which the Mericlone method is applied, for example, the following method has been proposed. The shoot apex of a plant belonging to the genus Wasabi of the Brassicaceae family is grown on a medium containing a plant growth hormone under light irradiation for 0.5 to 10 r.
A method for mass-producing wasabi seedlings, comprising spin-culturing at a rotation speed of pm, and culturing the obtained shoot primordium by stationary culture to produce seedlings (JP-A-61-115417).

[0007] The growth point site of wasabi is cultured in an adventitious bud induction medium containing, for example, a cytokinin-like plant growth regulator and a gibberellin-like plant growth regulator to obtain adventitious buds, and then the adventitious buds are auxinically planted. A method for growing wasabi seedlings, comprising culturing and rooting in a rooting induction medium containing a growth regulator and a gibberellin-like plant growth regulator, and a medium kit used for the method (Japanese Patent Laid-Open No. 5-130815).

A method for cultivating wasabi, comprising culturing wasabi growing point tissue in a liquid medium to form a shoot, and then culturing the shoot on a solid medium to form multiple shoots and proliferate (Japanese Unexamined Patent Publication No. -62694).

On the other hand, as a method of growing wasabi seedlings, which is developed by a method other than the above-mentioned mericlone method, an embryo culture, an adventitious embryo culture, a flower stem culture, a hairy root culture, a callus culture, and the like can be mentioned. . To illustrate some of them, first, for example, the following methods have been proposed as those using the above-described embryo culture method, somatic embryo culture method, and the like.

[0010] Immature embryos or seed tissues of wasabi (Brassicaceae, Wasabi genus) are cultured under dark conditions in an artificial medium to induce and propagate somatic embryos via callus, or to directly extract somatic embryos from tissues. A wasabi multiplication method comprising inducing and proliferating, and then regenerating a wasabi individual from the obtained adventitious embryo (JP-A-1-98416).

In a method for producing wasabi seedlings by plant tissue culture, a method for producing wasabi seedlings comprising obtaining seedlings from plant tissues via embryoid bodies (Japanese Unexamined Patent Publication No.
No. 3-304922).

Other reports other than the above publication include Haruki Kazuhisa et al .: Adventitious embryo formation and plant regeneration of wasabi by tissue culture, Kinki Chugoku Agricultural Research Institute, 75, 66-70 (1988), Haruki Kazuhisa: Wasabi Production of somatic embryos, agricultural technology, 43
(12), 15-18 (1988).

As a method for mass-producing wasabi seedlings by utilizing the above-mentioned flower stem, hairy root, callus, etc., for example, the following method has been proposed. A plant tissue of the Brassicaceae belonging to the genus Wasabi is cultured at a low temperature of 20 ° C. or less using a callus induction medium containing kinetin, 2,4-D, and thiamine to induce and proliferate the callus. A method for increasing wasabi seedlings, comprising redifferentiating in a regeneration medium containing milk and transforming them into seedlings (JP-A-53-86333).
No.).

[0014] Agrobacterium rhizogenes ( Agrobacter)
ii. Rhizogenes ) is used as a seedling for somatic shoots having adventitious roots formed directly from the roots by culturing the hairy roots induced in a medium containing no plant hormones in the dark and then culturing under light irradiation. Method for Producing Wasabi Seedlings (Japanese Patent Application Laid-Open No. 63-94921)
No.).

A method of mass-producing wasabi seedlings using flower stems, comprising segmenting the flower stems of wasabi that has begun to elongate and cutting them into seedlings by cutting and growing them in culture soil (Japanese Unexamined Patent Publication No.
-190112).

[0016] In addition to the above publications, there are reports other than the above publications: Osamu Matsumoto: Mass Propagation of Wasabi Seedlings by Tissue Culture, Agriculture, Pesticides, Materials, Technology, 31 (11) 8, 18-
21 (1987), Matsumoto R. et al .: Mass reproduction in vitro by culture of wasabi flower stem and rhizome tissue, Kinki Chugoku Agricultural Res.
3, 22-27, (1987), Masataka Sakatani: Propagation of Wasabi by Tissue Culture, Nara Prefectural Forestry Exp., 19, 5-10
(1989).

As described above, a large number of methods for mass-producing wasabi seedlings have been proposed. However, in the method of passing through calli during the cultivation, mutation is highly likely to occur, and the inherent pungency and flavor of wasabi are raised. There is a drawback that seedlings are not always maintained, and the culture method using mericlone or adventitious embryo has the advantage of producing seedlings with little mutation, but several kinds of cultures until seedlings are obtained. There is a problem that the culture operation is complicated, for example, it has to go through steps, and the growth rate is low.

As described above, although various methods of mass-producing seedlings by tissue culture have been proposed for wasabi plants, seedlings produced by using these techniques can be produced by conventional cultivation methods or hydroponic cultivation methods. It must be cultivated to adult plants, etc., and the cultivation period is still long,
The drawback that there is a danger of sickness during cultivation and poor growth due to unseasonable weather has not been solved. In addition, most of the mass-proliferation methods using tissue culture described above relate to the technology of increasing the number of above-ground parts and thereby mass-producing seedlings. No examples are found.

[0019]

Under these circumstances, the present inventors have studied the wasabi multiplication technique by tissue culture based on the above-mentioned prior art, and We succeeded in developing a new technology for enlarging it, but we also worked diligently to develop new technology for enlarging the wasabi rhizome and increasing the content of sinigrin, a pungent ingredient. As a result of stacking, it was found that the intended purpose could be achieved by growing the wasabi rhizome by tissue culture for a predetermined period of time using a specific medium and culture conditions, thereby completing the present invention. .

That is, an object of the present invention is to provide a method for cultivating wasabi rhizome which can specifically enlarge the rhizome of wasabi.

[0021] The present invention also relates to a method for aseptically culturing wasabi plants so that wasabi rhizomes can be produced in a short period of time.
It is another object of the present invention to provide a method for cultivating a wasabi rhizome which can remarkably enlarge the rhizome and increase the sinigrin content of the rhizome.

It is a further object of the present invention to provide a method for producing an enlarged rhizome containing a pungent component peculiar to wasabi and which can be used as a wasabi material. In addition, according to the present invention, since the wasabi rhizome is subjected to aseptic environmental control for the enlarging culture of the wasabi rhizome, unlike normal cultivation methods, there is a danger of sickness during cultivation and poor growth due to irregular weather. Can be avoided.

[0023]

According to a first aspect of the present invention for achieving the above object, a shoot (foliage) of a wasabi plant obtained aseptically contains inorganic salts and vitamins. Then, cytokinin was added to a basic medium containing a carbon source, and a sulfur-containing compound was further added to plant the medium in a sulfur-enriched state. This was continuously cultured without cutting, and the rhizome was enlarged. And a method for cultivating a wasabi rhizome in an enlarged manner.

Another aspect of the present invention is the above-described method for enlarging and culturing wasabi rhizome, wherein the sulfur concentration is 80 mg / l or more. Further, another aspect of the present invention is a method for cultivating the above wasabi rhizome, wherein the sulfur-containing compound is a sulfate compound,
It is.

Another embodiment of the present invention is the above-mentioned method for enlarging and culturing wasabi rhizome, which comprises adding cytokinin at a concentration of more than 0.01 mg / l. Another embodiment of the present invention is the above-mentioned method for culturing wasabi rhizome, wherein the medium contains 2 to 8% by weight of a saccharide as a carbon source. Furthermore,
Another embodiment of the present invention is the above-described method for culturing wasabi rhizome, wherein the medium contains 3.5 to 6.5% by weight of a saccharide as a carbon source.

Next, the present invention will be described in more detail. As wasabi plants targeted in the present invention,
Sawa Wasabi ( Wasabiajaponica Mats)
um. ), Horseradish ( Armorasia rust)
icana Gaertn. ), Lily wasabi ( Wasa
via tenuis Matsum. ).

In the present invention, a shoot (foliage) of a wasabi plant is used as a planting material. The foliage of this wasabi plant is obtained, for example, by a growth point culture as described in Examples. Not only that, shoots (foliage) can be used, such as those obtained from flower stalks, hairy roots, calli, etc., those obtained through somatic embryos, shoot primordia, etc. Absent. The culture of shoots (foliage) of the wasabi plant is carried out by adding a cytokinin such as 6-benzylaminopurine to a basic medium containing inorganic salts and vitamins and containing a carbon source, and further containing a sulfur-containing compound. Is added to the medium, and the medium is inoculated in a medium supplemented with sulfur to carry out the culture under specific culture conditions.

In this case, the basic medium may be a general medium used for tissue culture, for example, MS (Muras).
hige & Skoog) medium, LS (Linsma)
ier & Skoog) medium, B5 medium, Nitsc
h & Nitsch medium or any other medium having the same effect can be used. Further, the shape of the medium is not limited to a solid medium containing agar or the like, and a liquid medium or the like can also be used. Specifically, for example,
Large elements (NH ₄ NO ₃ , KNO ₃ , CaCl ₂ .2H
₂ O, MgSO ₄ .7H ₂ O, KH ₂ PO ₄ )
/ 2, and sucrose concentration of 2%
-Skoog's modified medium with cytokinin such as 6-benzylaminopurine at a concentration of more than 0.01 mg / l, preferably 0.1-10 mg / l, and about 0.7% -0.
A solid medium to which about 8 agar is added is preferably used.

The cytokinins used in the present invention include, in addition to 6-benzylaminopurine, zeatin,
Those having cytokinin activity such as kinetin, 4-pyridylphenylurea, and 2-isopentenylaminopurine can be used. Further, as the type of the carbon source, sucrose is preferred, but not limited to sucrose, and sugars generally used in tissue culture, such as glucose and fructose, can be used. Further, the concentration of the saccharide is preferably 2 to 8% by weight, and particularly, a medium in which the concentration of the saccharide is adjusted to 3.5 to 6.5% by weight enlarges the rhizome of wasabi. The above is exemplified as being more preferable. Examples of the sulfur-containing compound used in the present invention include the following. Sulfur-containing inorganic compounds: K ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ , N
a ₂ SO ₄ , MgSO ₄ , FeSO _4, etc. Sulfur-containing organic compounds: sulfur-containing amino acids, allyl sulfide,
Benzyl sulfide and the like As the sulfur-containing compound, a sulfuric acid compound is considered to be particularly preferable because the components of the sulfur-containing compound are mainly taken in as SO ₄ in the process of producing sinigrin.
Then, for example, K ₂ SO ₄ is added at 300 mg / l to 120 mg / l.
In the medium to which 0 mg / l was added, it was confirmed that the sinigrin content increased in proportion to the amount added.

In the culturing, the above shoots (foliage) are planted in the above-mentioned medium, and the shoots (foliage) are continuously cultured without cutting, preferably at a temperature of 10 to 30 ° C. Continue to enlarge the rhizome. The above culture may be performed for more than one month, usually for about two months. In the present invention, it is important that the shoots (stems and leaves) are continuously cultured under predetermined temperature conditions without cutting to enlarge the rhizome.

According to the study of the present inventors, the content of the pungent component sinigrin per g fresh weight is remarkably reduced as the rhizome of wasabi grows (the weight of the rhizome increases). By adding the sulfur-containing compound constituting the above to the medium, the sinigrin content per g fresh weight could be increased. The rhizomes enlarged as described above contain sinigulin and have myrosinase activity, so that the rhizomes are used directly as wasabi material or the rhizomes as in the case of wasabi cultivated individuals. It is possible to extract the pungent component peculiar to wasabi by using the method.

It is not clear why the plant hormone cytokinin causes hypertrophy when it enters the plant body. However, in this regard, when the potato tuber is enlarged, the cytokinin-like activity in the tuber is increased. [Jameson P. E. FIG. et a
l. Physiol Plant, 63 (1), 53
-57 (1985); E. FIG. et a
l. , Nature, 221, 279-280 (196).
9)] However, no reports have been found on the enlargement of wasabi rhizomes, and the above findings were obtained for the first time by the present inventors.

[0033]

Next, the present invention will be specifically described based on examples, but the present invention is not limited to only the examples. Example 1 (1) Extraction of a growth point site In this example, a daruma-based wasabi (Izumi Daruma) from Shizuoka was used.
This was performed using The rhizome was cut into pieces at a distance of 1 to 2 cm from the base of the petiole of the rhizome, and the outer skin and the upper part of the petiole were cut off. The rhizome was immersed in a 4% available chlorine sodium hypochlorite solution for 15 minutes and then rinsed with sterile water. The side buds at the base of the petiole of the rhizome and the vicinity of the growing point sterilized in this way were placed in a clean bench at 0.5 m under a stereoscopic microscope.
It was extracted to a size of m to 2.0 mm.

(2) Cultivation of the growth point site Large elements (NH ₄ NO ₃ , KNO ₃ , CaCl ₂ .2H
₂ O, MgSO ₄ .7H ₂ O, KH ₂ PO ₄ )
/ 2, and sucrose concentration of 2%
& 0.1 concentration of 0.1 mg / l in modified medium of Skoog
15 ml of benzylaminopurine plus 0.7% agar was placed in a test tube, and one growth point site extracted as described above was planted per test tube. The cultivation was performed at a temperature of 14 ° C. under irradiation with a fluorescent lamp at about 3,000 lux for 12 hours. Two months after the start of culture, shoots (foliage) were formed.

(3) Enlargement culture of rhizome The shoots (foliage) obtained as described above were subcultured in a 200 ml culture bottle containing 40 ml of a medium having the same composition. (Foliage) was cut so that the number of leaves was 3 to 5. The sucrose concentration was 3.5% and the 6-benzylaminopurine concentration was 5%.
Murash, which was added to 1200 mg / l of K ₂ SO ₄ to adjust the sulfur concentration to 252.24 mg / l.
image &Skoog's modified medium, about 3,0
The culture was performed at a temperature of 20 ° C. under a fluorescent lamp irradiation at 00 lux for 12 hours. The weight of the rhizome at the start of the culture was 0.02 g. When the culture was continuously performed for 2 months without cutting the shoots, the rhizome part was remarkably enlarged, and the weight of the rhizome became 0.46 g, which was increased about 23 times in the culture for 2 months. The sinigrin content was 688.54 μg.
/ G fresh weight.

Comparative Example As a comparative example, (1) and (2) of Example 1 were carried out in the same manner, and K ₂ SO ₄ was not added to the medium (3) for planting shoots (foliage). Others are Example 1.
A similar experiment was performed in the same manner as described above. Continuously in medium without K ₂ SO ₄ (sulfur concentration 31.44 mg / l)
When the rhizome was cultured for months, the rhizome was enlarged to the same extent as in Example 1 and weighed 0.46 g, but the sinigulin content was 43.06 μg / g fresh weight, and the rhizome of Example 1 was sinigrin. The level was as low as about 1/16 of the content.

Comparative Test Results Evaluation test of the effect of sulfur concentration The effect of the sulfur concentration on the rhizome sinigrin content of the tissue culture seedlings of wasabi was determined in the same manner as the culture method and culture conditions in Example 1 except that the concentration of K ₂ SO ₄ was changed. Tested. Table 1 shows the results. From the results apparent Table _1, the K 2 SO ₄ 300mg / l~120
0 mg / l to reduce the sulfur concentration from 86.64 mg / l to 2
By setting the concentration to 52.24 mg / l, the content of sinigrin in the enlarged rhizome was remarkably increased, and the content was increased by about 4.4 to 16.0 times as compared with the case where K ₂ SO _{4 was} not added.

[0038]

[Table 1]

2. Evaluation test of pungent component About using wasabi tissue culture as a wasabi raw material, a method for producing wasabi-like flavor from wasabi callus has already been proposed, but the present invention relates to wasabi tissue culture seedlings. It is clearly different from the prior art in that the rhizome is subjected to hypertrophy culture and the enlarged rhizome is used as a wasabi material. In order to verify the superiority of using the enlarged rhizome as a wasabi raw material, the present inventors conducted an evaluation test on the sinigulin content and myrosinase activity of the rhizome of the tissue culture seedling of wasabi, the callus and the rhizome of the mother plant. As a result, it was found that the calli did not contain sinigrin, but the rhizomes of the enlarged tissue culture seedlings contained sinigrin and had myrosinase activity. Also, the rhizome is
It turned out that when grated with a grater, it shows pungency.

As described above, the rhizome of the wasabi tissue-cultured seedling of the present invention contains sinigulin and has a myrosinase activity, and thus can be considered as a wasabi raw material closer to the rhizome of the mother plant than the callus. In general, the sinigrin content varies depending on the variety, the cultivation place, the harvest time, and the fertilization conditions. In the conventional literature, the sinigrin content (μg /
g fresh weight) is described in, for example, Riki Furuya: Plant Tissue Culture, 5
According to (2), 82-86 (1988), it is 492 in the case of the rhizome of the mother plant. According to 16, 38-43 (1985), it is 1300 to 7200 in the case of the rhizome of the mother plant.

Further, the rhizome of the tissue culture seedling of wasabi wasabi according to the present invention is biologically a rhizome and not a callus. That is, the enlarged rhizome obtained by the method of the present invention has the same cambium and vascular structure as the rhizome of the mother plant, and can be said to be basically the same as the rhizome of the mother plant. Without such a structure, it was found that the enlarged rhizome of the tissue culture seedling according to the method of the present invention was not callus.

[0042]

As described above in detail, the present invention provides a method of culturing wasabi plants by culturing wasabi in a medium having the same composition for a predetermined period of time without using a specific medium and culture conditions. The present invention is characterized in that the rhizome is enlarged. According to the present invention, the following effects can be obtained. (1) The rhizome of a wasabi tissue culture seedling can be enlarged in a short period of time, and the sinigulin content of the rhizome can be significantly increased. (2) Since the enlarged wasabi rhizome obtained by the present invention contains a component related to the pungency of wasabi, it can be used as a wasabi material similarly to a cultivated individual of wasabi. (3) In addition, since the enlargement of the rhizome of wasabi is carried out under a sterile control environment, it is possible to avoid dangers such as disease during cultivation and poor growth due to unseasonable weather.

Claims (6)

(57) [Claims] 1. A shoot (foliage) of a wasabi plant obtained aseptically is prepared by adding cytokinin to a basic medium containing inorganic salts and vitamins and containing a carbon source, and further adding a sulfur-containing compound. A method for cultivating a wasabi rhizome, wherein the rhizome is enlarged by planting the medium in a medium supplemented with added sulfur and continuously cultivating the medium without cutting. 2. The method according to claim 1, wherein the sulfur concentration is 80 mg / l or more. 3. The sulfur-containing compound is a sulfuric acid compound.
The method for hypertrophic culture of wasabi rhizome according to the above. 4. The method of claim 1, wherein the cytokinin is added at a concentration of more than 0.01 mg / l. 5. The method according to claim 1, wherein the medium contains 2 to 8% by weight of a saccharide as a carbon source. 6. The medium according to claim 3, wherein the saccharide is used as a carbon source in an amount of 3.5 to 3.5.
The method for enlarging and cultivating wasabi rhizome according to claim 1, which contains 6.5% by weight.