KR20060113156A - Method for mass-producing wasabia and its root - Google Patents

Abstract

A method for mass-producing wasabia and roots thereof is provided to stably and inexpensively produce the wasabia and roots thereof within short period of time by using a bio-reactor and optimizing culture conditions. The method comprises the steps of: (a) cutting meri-stem of aseptically tissue-cultured wasabia plant and then proliferating it in a solid medium such as woody plants medium(WPM) containing carbon source such as sucrose and maltose, cytokinin and agar; and (b) growing the proliferated wasabia plant in a liquid medium containing carbon source and cytokinin.

Description

Mass production method of wasabi and wasabi root {METHOD FOR MASS-PRODUCING WASABIA AND ITS ROOT}

1 is a photograph showing 'dalmatian' varieties of horseradish W asabia japonica Matsum used as a plant material in an embodiment according to the present invention.

2a to 2b is a photograph showing the plant (Fig. 2a) and its root (Fig. 2b) used in the liquid culture of wasabi and wasabi root according to the present invention.

Figure 3 is a photograph showing the effect of plant growth according to the type of cytokinin (from the left benzylaminopurine, kinetin, thidiazuron, zitin).

Figure 4 is a photograph showing the growth of wasabi when the concentration of indolebutyric acid (IBA; 3-indolebutyric acid) and benzylaminopurine (BA: 6-benzylaminopurine).

5 is a photograph showing the growth of wasabi according to the concentration of the carbon source.

Figure 6 is a photo showing the culture of the plant using a bioreactor (left side culture on the surface, the middle and right side of the culture in the surface).

FIG. 7 is a photograph showing horseradish plants cultured in the bioreactor sleeping layer (left) and in the water surface (right), respectively.

Figure 8 is a photograph showing the root growth of horseradish after single treatment of indole butyric acid (IBA) and naphthylacetic acid (NAA).

9A to 9C are photographs showing in-flight culture plants (FIG. 9A), in-flight plant roots (FIG. 9B), and purified plants (FIG. 9C) used as experimental materials for cultivation conditions.

10a to 10e are photographs showing the appearance of the wasabi during the hydroponic cultivation (Fig. 10a, 10b, 10c) and harvesting (Fig. 10d, 10e).

11A to 11C are photographs showing the culture of horseradish roots using an air-lift method in a bioincubator.

The present invention relates to a method for mass production of wasabi and wasabi root, and more particularly to a method for mass production of wasabi and its root in a disease-free state stably and inexpensively through tissue culture.

Wasabi is a perennial ripening plant that grows in Japan and Korea's Ulleungdo, and grows in halftones of mountain valleys where cool and clear water flows. It has a spicy, sweet and fragrance, and is mainly used as a spice for sashimi, sushi, and noodles. Allyl isothiocyanate, which is present in the roots of horseradish components, promotes digestion, promotes the synthesis of vitamin B ₁ , inhibits the oxidation of vitamin C, inhibits abnormal fermentation after eating, insect repellent, antibacterial, antifungal, anticancer, It is known that there is an action such as insecticide.

Recently, due to the increasing consumer's health needs, attention to natural preservatives instead of artificial food preservatives with safety problems such as residual toxicity or mutagenesis has been increasing, horseradish or its active ingredient has attracted more attention.

In addition to the above uses, natural pesticides, antibacterial soaps, fragrances, room fresheners, deodorants using horseradish or its active ingredients have been developed in Korea, and in Japan, alcohol, tea, laundry cleaners, paper, labels, It is also utilized in films.

As described above, wasabi has a high degree of utilization, and although consumption is increasing gradually, cultivation is difficult to meet domestic demand. Even so, the amount used in restaurants is very short, and the domestic use of horseradish imitation products imported from Japan or mixed with pigments is mainly used.

Wasabi is a very sensitive crop, which is difficult to cultivate due to its narrow growth range. In addition, wasabi must be grown for more than one year in order to be a commodity. Cultivation is difficult and difficult due to the fact that it can only be cultivated in regions where the climate is cool throughout the year, the temperature difference is small, and clear water is always flowing.

In addition, in the early stages of cultivation of horseradish, Korea is mainly using live breeding, which lacks the seeding system and is cultivating a considerable amount of seeded as expensive seeds imported from Japan. Seeds do not withstand dryness, deep dormancy, low germination rate, uneven growth, and cross-hybrid, so it is difficult to maintain superior traits in the latter by seed propagation. Some farms are cultivated by self-harvesting, but deterioration of superior traits is severe.

Even if a good individual is selected, the seeding rate is low by the existing abandonment and dividing method, so that about 10 weeks per year are grown. After selection, it takes a lot of time to grow large quantities, so it is difficult to supply the seedlings cheaply because it cannot meet the rapidly increasing demand and the production cost is high.

In addition, horseradish is generally used for propagation using seedlings branched from the seedlings as seedlings. Such a method causes transmission of viruses to newly produced individuals, causing a decrease in production volume. In other words, the production method using abandonment division degenerates after several generations, and thus, continuous dissemination of disease free bottles is necessary.

In addition, in the case of wasabi, there are still problems such as quantitative shortage of materials due to contamination and difficulty in collecting pesticides during cultivation, since they are mostly grown on forge or used as raw materials for processing.

Therefore, the necessity of establishing mass harvesting of high quality seed cultivation and high quality multiple cultivation techniques by tissue culture or the like has emerged.

Due to this necessity, research on mass production technology of wasabi through tissue culture has continued in Japan and Korea. Generally, wasabi is tissue cultured in vitro, and after it has been purified outside, it is grown in water or field.

At this time, the solid medium is used when culturing in the cabin, the solid medium, there is a disadvantage that takes a lot of labor during subculture.

Purified wasabi is cultivated through water cultivation or field cultivation, but water cultivation is cultivated in wetlands of mountain valleys, so the cultivation site is limited, packaging costs are high, and the cultivation period takes 18 months or more. There is this. In addition, in the case of field cultivation, there is a disadvantage that the quality is degraded, the growing period takes about 14 months.

Therefore, there is an urgent need to develop a mass production technology that can supply horseradish at a lower cost and in large quantities.

In order to solve the above problems, the present invention provides a method for mass production of wasabi and its roots in a short and stable time by optimizing the culture conditions while using a bioreactor capable of mass replication of plants. There is that purpose.

In order to achieve the above object, the present invention comprises the steps of propagating in a solid medium containing a carbon source, cytokine (cytokinin) and agar components by cutting the apex of the aseptic tissue cultured horseradish plant; And it provides a wasabi was produced in a mass production method and wasabi produced by the wasabi plant grown in the solid medium in a liquid medium containing a carbon source and cytokinin.

The present invention also comprises the steps of inducing the roots in a solid medium containing a carbon source, auxin and agar by cutting the apex of the horseradish plant cultured aseptically; And it provides a mass production method of wasabi root and wasabi root produced by culturing the wasabi root derived from the solid medium in a liquid medium containing a carbon source and auxin to grow the root.

In addition, the present invention provides a horseradish active ingredient extracted from the wasabi and wasabi root produced above.

Thus, according to the present invention, it is possible to cultivate wasabi seedlings or horseradish roots produced by tissue culture in a bioreactor, it is possible to provide wasabi biomass (biomass) on an industrial scale will be.

Hereinafter, the present invention will be described in more detail.

First, the present invention comprises the steps of propagating in a solid medium containing a carbon source, cytokine (cytokinin) and agar components by cutting the apex of the horseradish plant cultured aseptically; And it provides a mass production method of wasabi horseradish wasabi grown in the solid medium in a liquid medium containing a carbon source and cytokinin.

The medium that can be used in the growth step and growth step is not particularly limited as long as the medium that can be used for plant culture, among them, WPM (Woody Plants Medium) medium is preferred. In the case of WPM medium, for example, the concentration may be adjusted to x1, x1 / 2, x1 / 4, x1 / 8, or the like, but the lower the WPM concentration used for the medium, the smaller the leaf area and the leaf color. As it tends to fade, it is preferable in terms of plant growth to use it at its original WPM medium concentration without dilution.

The solid medium and the liquid medium used in the growth and growth steps are preferably used by adjusting the pH to 4.5-9, more preferably 4.8-7.

The solid medium used in the propagation step includes a carbon source, cytokinin and agar, but at this time, the carbon source is not particularly limited, but sucrose or maltose ( maltose) is preferably used. The concentration of these carbon sources is preferably 2-9%.

The cytokinin contained in the solid medium is not particularly limited if it can induce aerobics, among which benzylaminopurine (BA), thidiazuron (TDZ: thidiazuron), kinetin and Jia At least one substance selected from the group consisting of tin is preferred, and more preferably benzylaminopurine (BA) is used alone. At this time, the concentration of cytokinin that can be used is preferably 0.01 ~ 1mg / L.

The solid medium used in the propagation step may further include auxin, and the auxin that can be used at this time is not particularly limited as long as it can induce the root of wasabi, indole butyric acid (IBA) Is preferred. Thus, when cytokinin and auxin are mixed, the amount of cytokinin contained in the solid medium is preferably 0.01 mg / L, and the amount of auxin is 0.01-1.0 mg / L.

It is said that there is no particular limitation as an agar component contained in the solid medium, among which agar or gelrite is preferred, and in particular, 0.15% of gelite is most preferred.

In the proliferation step, it is preferable to incubate for 1-2 months under conditions of 7-9 hours at a temperature of 10-20 ° C.

In the growth stage of wasabi is preferably cultured using a liquid medium in a bioreactor or a culture vessel, it is particularly preferable to culture wasabi plants in the water surface of the liquid medium. In addition, when cultivating wasabi plants in a bioreactor, there is no particular limitation, and among them, it is preferable to use the ebb and flood or the air-lift method.

The liquid medium in the growth stage includes a carbon source and cytokinin, and at this time, there will be no particular limitation on the carbon source, but sucrose or maltose is used. It is preferable. The concentration of these carbon sources is preferably 2-9%, most preferably sucrose 2-9%. In addition, there are no particular limitations on the cytokinin which can be used, but among them, benzylaminopurine (BA), thidiazuron (TDZ), kinetin and zeatin (Zeatin) At least one substance selected from is preferred, more preferably benzylaminopurine (BA) is used alone. At this time, the concentration of cytokinin that can be used is preferably 0.01 ~ 1mg / L, most preferably to use benzylaminopurine (BA) 1mg / L.

In the growth stage of wasabi plants, it is preferable to incubate wasabi plants for 1-2 months in a dark state at a temperature of 10-20 ℃.

As described above, in the case of the present invention, it is possible to mass-produce horseradish disease-free seedlings in a short period of time by growing wasabi in solid medium and growing wasabi plants in a liquid medium in a bioreactor or culture vessel.

By using the wasabi-free seedlings so obtained, in order to cultivate wasabi in large quantities, a purification step, nutrient solution cultivation may be further added. That is, horseradish plants that have undergone growth stages in the bioreactor are taken out of the air and subjected to a purification stage. The purifying step is preferably performed under conditions of a temperature of 10-20 ° C in a mixture of vermiculite and perlite in a ratio of 7: 3.

Wasabi plant after the purification step in the above can be cultivated after nutrient solution. The nutrient solution cultivation may be made by using any one medium selected from the group consisting of hydroponic cultivation, solid medium single (pearlite) and solid medium mixed (pearlite: pitmos = 7: 3).

The present invention also provides wasabi produced by the mass production method of the wasabi.

Furthermore, the present invention provides a method for mass-producing only a portion of wasabi for the purpose of mass-producing a specific effective ingredient of wasabi, in particular, by cutting the apical part of wasabi cultured aseptic tissue source carbon source, auxin And inducing roots in a solid medium containing agar components; And it provides a mass production method of horseradish root comprising the step of propagating the root by culturing the wasabi root derived from the solid medium in a liquid medium containing a carbon source and auxin.

The medium that can be used in the growth step and growth step is not particularly limited as long as the medium that can be used for plant culture, among them, WPM (Woody Plants Medium) medium is preferred. In the case of WPM medium, it is preferable to use the original concentration without dilution in terms of root growth.

The solid medium and the liquid medium used in the growth and growth steps are preferably used by adjusting the pH to 4.5-9, more preferably 4.8-7.

The solid medium used in the propagation step includes a carbon source, auxin and agar, but at this time, there is no particular limitation on the carbon source, but sucrose or maltose is preferably used, and these carbon sources The concentration of is preferably 2-9%.

As the auxin that can be used in the solid medium, if there is no particular limitation as long as it can induce the root of wasabi, indolebutyric acid (IBA) is preferred. The amount of auxin contained in the solid medium is preferably 0.01-1 mg / L.

It will be said that there is no particular limitation as an agar component contained in the solid medium, among which agar or gellite is preferred, and in particular, 0.15% of gelite is most preferred.

In the propagation step, it is preferable to incubate for 1-2 months at a temperature of 10-20 ° C. under conditions of 7-9 hours.

When the horseradish roots grown in the solid medium are inoculated into the liquid medium, the root length thereof is preferably 1-2 cm, more preferably 1.2-1.7 cm.

In the growth stage of wasabi root is preferably cultured using a liquid medium in a bioreactor or culture vessel, in this case, it is particularly preferable to culture wasabi root in the surface of the liquid medium. In addition, when cultivating horseradish roots in a bioreactor, there is no particular limitation, and among them, it is preferable to use the ebb and flood or air-lift method.

The liquid medium in the growth step includes a carbon source and auxin, wherein the carbon source is preferably sucrose 2-9%. In addition, the auxin is preferably indolebutyric acid (IBA), and the concentration of the auxin used is preferably 3-5 mg / L.

In the growth stage of wasabi root, it is preferable to incubate wasabi root for 1-2 months in a dark state, temperature 10-20 ℃.

The present invention also provides horseradish roots mass-produced by the above method.

In addition, the present invention provides a horseradish active ingredient extracted from wasabi plant and wasabi root mass-produced by the above method.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the scope of the present invention is not determined accordingly, but is presented by way of example only.

[ Example]

Example  1: horseradish plant of  massive production

1) Growth stage

Plant material was used as a plant was grown by aseptically disinfecting 'dalmatian' varieties (Fig. 1) in wasabi W asabia japonica Matsum and introduced into the cabin.

To disinfect the material, the soil on the leaves, stems, and roots was shaken off and soaked in running tap water for about an hour. The material was then transferred to a clean room, surface sterilized with 70% ethanol for 10 seconds, and washed three times with sterile water. The solution was immersed in 2% of sodium hypochlorite solution (Lax) for 15 minutes, sterilized, rinsed 3-5 times with sterile water, and the sample was dried using sterile filter paper. From these materials, growth points, including foliar phases, were removed and placed on WPM basal medium to obtain complete plants and passaged to obtain a large number of aseptic plants.

In the growth stage using the solid medium, the basic medium was WPM (Woody Plants Medium) medium, and 3% sucrose and 0.15% of gelrite were added, and the pH was adjusted to 5.7. The amount of the medium was dispensed in a square container (made by Vitrosys Co., Ltd.) at 150 ml each, and 1 mg / L of benzylaminopurine (BA) was added to the medium to induce multiple shoots in the early plants grown at the growth point including the foliar phase. The apical part to which a number of derived plants were connected was separated with a knife and used as an experimental material. The medium was dispensed into a square container (manufactured by Vitrosys, Inc.) and incubated for 2 months at a temperature of 18 ° C. for 8 hours.

2) growth stage

The entire sterile plant subcultured to the solid culture in the growth step was used for the experiment (Fig. 2a).

The basal medium was added with sucrose 3% using WPM medium and the pH was adjusted to 5.7. The amount of medium was cultured with 3L in 5L bioreactor and 5L in 10L bioreactor to culture the whole plant with leaves and roots. The culture of the bioreactor is performed by continuously injecting the culture solution into the ebb and flood method or the bioreactor, in which the culture medium stays in the bioreactor for a certain period of time, and then repeats the process of flowing out to an external storage tank. An air-flow method was used. In order to induce multiple shoots, 1 mg / L of benzylaminopurine was added, and cultured for 1 month in a dark state at 18 ° C.

3) Purifying stage

The topsoil mixed with vermiculite and perlite in a ratio of 7: 3 was placed in the growing box, and the cultured plants were taken out of the bioreactor and planted. Purifying conditions were to maintain the relative humidity of 95% or more at a temperature of 18 ℃ to be shaded.

4) Nutrient cultivation stage

The purified wasabi was mixed with pearlite and peat moss, and the nutrient solution was cultivated by drip irrigation four times a day to obtain a large amount of wasabi seedlings.

Example 2 Growth and Growth of Wasabi Roots

1) Growth stage

As auxin instead of cytokinin, indolebutyric acid (IBA) was used to grow wasabi roots in the same manner as in Example 1 except that the roots were induced.

2) growth stage

In the propagation step, only the root portion of the aseptic horseradish plant passaged as a solid culture was used for the experiment (Fig. 2b).

The basal medium was added with sucrose 6% using WPM medium and adjusted to pH 5.7. The amount of medium was cultured by cutting only roots by dispensing 3L in 5L bioreactor and 5L in 10L bioreactor. For root proliferation, 3 mg / L of indolebutyric acid (IBA) was added and cultured. It was cultured for 1 month in a temperature of 18 ℃, dark, to obtain a large amount of wasabi root (Figs. 11a to 11c).

Experimental Example

Experimental material

As in the above embodiment, sterilized sterilized horseradish apex was cut and transferred to a solid medium to which 0.5-1 mg / L of benzylaminopurine (BA) was added, followed by culturing for 1 month in multiple shoots. The apical part including the solution was used as the material in the following experimental example.

However, in the case of hormone-related experiments, in order to minimize the effect of benzylaminopurine, one generation used plants that were incubated without adding benzylaminopurine to the WPM medium before the experiment.

Experimental Example 1 Investigation of Liquid Growth Promoting Effect According to Cytokine Kinds

The reaction of horseradish plants according to the type of cytokinin, a plant growth regulator that induces cell division and differentiation of malaria, was examined (Table 1).

Experimental results showed that 0.1 mg / L treatment of thidiazurone (TDZ) was 11.1 and 40.9 leaves, respectively, compared to 2.1 control and 10.5 leaves of the control, which were the most effective in promoting the growth of liquid embryos compared to other cytokinins. there was. However, the plant morphology and color of the tidiazuron treatment were malformed.

From Tables 1 and 3 below, it was considered that benzylaminopurine treatment was most suitable for promoting liquid growth and leaf growth.

Experimental Example 2 Comparison of Effects of Benzylaminopurine (BA) and Indolebutyric Acid (IBA) on the Formation of Abnormal Child and Root

To investigate the effects of cytokinin benzylaminopurine and auxin type indolebutric acid on multiple shoot induction and root formation, concentrations (0, 0.1, 0.5, 1, 2mg each) Benzylaminopurine and indolebutyric acid were added singly or in combination. Addition of benzylaminopurine increased the number of aliquots and leaves, shortened herb length, and shorter leaves due to smaller leaves (Table 2). Benzylaminopurine has an excellent effect on the development of liquids, and the benzylaminopurin untreated group has about 1.2 aliquots, whereas the higher the benzylaminopurine concentration, the more the number of aliquots and leaves. (FIG. 4). As a result, it was judged that benzylaminopurine 0.5-1mg / L concentration would be suitable for plant growth aimed at securing population.

Based on the results of Table 2, the re-test was performed by lowering the concentration of benzylaminopurine than Table 2 in order to match the concentration ratio of cytokinin and auxin that simultaneously meets plant growth and root induction (Table 3).

From the results of Table 3, the concentrations of benzylaminopurine and indolebutyxide obtained simultaneously in proliferation and root induction during in-situ solid culture were the highest in the indolebutyric acid 0.1-0.5mg / L in 0.01 mg / L of benzylaminopurine. It was found to be effective.

Experimental Example 3: Comparison of the Effects of Types of Colloids and WPM Concentrations on Liquid and Root Formation

Plant growth according to agar and gelite treatment, a colloidal material that plays a role of supporting plants, was compared (Table 4). Compared with the agar, the number of leaves of the gelite treatment and the root growth were long and good. In addition, the use of gelite was good for increasing the live weight, which is about twice the total live weight.

On the other hand, the lower the WPM concentration in the medium, the smaller the leaf area and the lighter the leaf color, the brighter the greenish green color. In other words, using the original WPM medium concentration without dilution was the best for plant growth.

Experimental Example 4 Comparison of Growth According to Sucrose Concentration

Growth of wasabi was compared according to the concentration of sucrose in the medium. The results are shown in Table 5 below.

As a result, the lower the sucrose concentration, the more leaves, the longer the grass length, and the root number or weight was better at 6% than 3% (Table 5). In terms of appearance, 3% of sucrose leaves were dark green, and 6% of the treatments showed some browning, and the leaf veins developed more than 3% of treatment and brighter, but the roots were the best. Highest (Figure 5). On the other hand, as the sucrose concentration was higher than 9%, the stems were bent, the leaves and stems became harder, and the leaves were dried due to the progressive color of the leaves.

Experimental Example 5: Changes in Liquid Solution and Root Formation According to pH

Changes in solution formation and root formation with pH change in the medium were investigated. The results are shown in Table 6.

As a result, the state of pH 5 treatment was dark green. The pH 7 treatment was pale green and the leaf edge was pale green.

Root water was the most at pH 5 treatment, but there was no significant difference in root weight, so experiments were conducted to subdivide between pH 5-7 (Table 7).

As a result, the lower the pH, the higher the number of roots and the higher the live weight.

Experimental Example 6: Comparison of Wasabi Plant Growth According to Carbon Sources

Growth of horseradish plants according to carbon source was compared. The results are shown in Table 8 below.

As a result, the number of leaves, leaf color, root number, and root of sucrose treatment were good, but maltose treatment was the highest in herb, leaf and whole plant live weight. On the other hand, the fructose treatment showed a black discoloration at the base of the leaf disease and root rooting, but the length was short and short. Sucrose treatment was considered most optimal in terms of growth and price.

Experimental Example 7: Comparison of Root Formation According to Indolebutyric Acid Concentration in Medium

When using it as a seedling, roots did not form when benzylaminopurine was treated, so the survival rate was reduced when transplanted to soil (data not shown). Therefore, after securing a large number of seedlings with benzylaminopurine, it is thought that a method of inducing rooting by inducing rooting and transplanting it in vitro is preferable. In order to increase the root formation rate, the root formation rate according to the indolebutyric acid concentration in the medium was investigated (Table 9).

In the results of Table 1, when the concentration of indolebutyric acid was high, unlike root formation, when the concentration of indolebutyric acid was set to 0.01-1 mg / L, all rooting was performed. Among them, it was found that 0.01 mg / L of indolebutyric acid, which has a large number of leaves and long leaves, is suitable for use as a rooting medium.

Experimental Example 8: Comparison of growth of horseradish plants during solid culture and liquid culture using bioreactor

3% sucrose was added, 2 L of WPM medium adjusted to pH 5.7 was added to a 5 L bioreactor, and 100 plants were inoculated, and then cultured for 30 days, and compared with the solid culture plants. In particular, in order to compare the growth according to the culture method in the culture of the bioreactor, the ebb and flood method and the air-lift method was compared and the results are shown in Table 10.

In the cultivation method, EB and flood was slightly higher in stem elongation, root growth, and total biomass than the air-lift method, but in the case of EB and flood, the air itself is more complicated in the industrialization process. Appropriate.

In addition, the air-lift bioreactor was cultured by dividing it into a method of culturing in the sleeping layer and a method of culturing in the water surface. The results are shown in Table 11 and FIG.

After 10 days of culture, the plants circulated by air supply floated on the surface of the medium, and in the early stage of culture, the tissues absorbed a lot of water and showed a form of overhydration. It began to grow (Figures 6, 7). Sleep layer cultures showed blackening of the leaf basal area and no elongation of the roots. After 25 days of incubation, the plants were facing the light and the roots were in good condition. After 30 days of incubation, the plant live weight of the submerged culture treatment group was 4.84 mg, which is about 6 times higher than the 0.8 g of the solid culture, and the bioreactor liquid culture was effective for plant growth.

Experimental Example 9: Effect of Auxin Type and Concentration on Growth and Growth of Wasabi Root

Experiments were conducted to find the optimum culture conditions for wasabi root culture. First, roots cut from plants were mixed with different concentrations of indolebutyric acid (IBA) and naphthylacetic acid (NAA), which are plant growth regulators (PGRs), which are involved in root elongation. Root growth was observed after incubation. The higher the indolebutyric acid treatment concentration, the longer the root length and the more proximal occurrence, whereas the higher the naphthylacetic acid treatment concentration, the higher the incidence of the extensors but the elongation of the kidneys. High (Table 12). In addition, in the control group that was not treated with plant growth regulators, the root length of the root was well elongated, but no geodes were formed. Therefore, it can be seen that the indolebutyric acid treatment is effective for root culture.

(Unit: mg / L-1) IBA 0 IBA0.5 IBA 1 IBA 3 IBA 5 NAA 0

NAA0.5

NAA 1

NAA 3

NAA 5

In addition, when naphthylacetic acid treatment concentration is lower than indolebutyric acid treatment concentration when the indolebutyric acid and naphthyl acetate extract are mixed, the number of extensors is the same as that of naphthylacetic acid treatment. The roots of were short and wasabi was sensitive to naphthylacetic acid. In the wasabi experiment, the effect of increasing the root growth rate by the mixed treatment was not noticeable. Therefore, the mixed treatment is not suitable for root proliferation, and it is considered that it is preferable to treat only indolebutyric acid alone, and the re-test was performed by changing the concentration of indolebutyric acid (Table 13).

In the case of indolebutyric acid treatment, the roots of the roots developed a new geodetic surface at the root surface, and there was a large number of extensors during naphthylacetic acid treatment, but the length was not elongated and rapidly aging and browned. 8).

In addition, it can be seen from the results of Table 13 that the indolebutyric acid treatment concentration of 3-5 mg / L is most effective for root growth.

Experimental Example 10: comparison of root growth according to the type of carbon source

As shown in Table 14, as a result of observing the root growth by different carbon source, glucose, maltose, fructose treatment all browned roots, proliferation rate is reduced and sucrose treatment When compared to other treatments, the growth rate was more than three times.

From the results in Table 14, it was found that sucrose was most effective in wasabi root growth.

Experimental Example 11: comparison of root growth according to the concentration of sucrose

When the sucrose 2%, 3%, 6% and 9% were respectively treated to determine the effect of the sucrose concentration, as compared to wasabi root growth, the higher the sucrose concentration, the higher the live weight. Overgrowth tended to increase (Table 15). This was because 2% and 3% treatments had only one root, but 6% and 9% treatments showed secondary root growth. In terms of price, 6% sucrose was considered the most appropriate.

Experimental Example 12: Comparison of growth rate according to root length when inoculated into liquid medium

Changes in growth were investigated by varying the root length inoculated in root culture (Table 16). When the root length was 0.2cm at the time of inoculation, the root length was 1.5cm when harvested and 2.7cm when incubated at 1.5cm. Based on these results, the shorter the root length at inoculation, the higher the elongation rate. However, comparing the biomass with the growth rate, the longer the root length at the time of inoculation, the higher the biomass weight was found to be most suitable when the root length at about 1.5cm.

Experimental Example  13: Outside  Plants according to cultivation conditions Live weight  change

In order to establish a wasabi plant production method that can replace water cultivation and field cultivation, which is a general cultivation method of wasabi was taken out of the air using a plant grown in the cabin and purified (Fig. 9a to 9c) in the nutrient solution under various conditions Wasabi growth differences were investigated.

Treatment medium forms were hydroponic cultivation, solid medium single use (pearlite), solid medium mixed (pearlite: pitmos = 7: 3), and hydroponic cultivation used solid culture and liquid cultured plants in the aircraft (Figs. 10A to 10D). Purified horseradish plants were incubated in the cabin, and hydroponic cultivation and solid nutrient cultivation were carried out. As a result, the leaf area and volume of the treatment group mixed with pearlite and peat moss and drip irrigation four times a day (without data) showed the highest weight of the plant (Table 17).

As described above, according to the present invention, by growing the wasabi through liquid culture in the bioreactor, it is possible to stably produce a wasabi, mass production at a low cost, and to reduce labor.

In addition, according to the present invention, it is possible to mass-produce horseradish plants in a bioreactor, so that the diseases inherent in the roots can be infected through nutritional propagation, and new varieties are propagated in a short time, and viruses in old varieties It can be removed.

Furthermore, in the case of the present invention, by providing a method for cultivating wasabi seedlings grown in-flight after cultivation, not only convenient, but also mass production in a short time compared to conventional methods for growing wasabi by field cultivation, water cultivation This becomes possible.

In addition, it is possible to mass-produce not only horseradish plants, but also specific parts, especially roots thereof, thereby mass extracting the necessary active ingredients therefrom.

Claims (28)

Cutting the apex of the aseptic tissue cultured horseradish plant and propagating in a solid medium containing a carbon source, cytokinin and agar; And A method for mass production of wasabi comprising growing wasabi plants grown in the solid medium in a liquid medium containing a carbon source and cytokinin. The method of claim 1, The medium is a mass production method of wasabi was WPM (Woody Plants Medium) medium. The method of claim 1, The medium is a mass production method of wasabi was adjusted to pH 4.8-7. The method of claim 1, The carbon source is sucrose or maltose mass production method of wasabi. The method of claim 1, The concentration of the carbon source is 2-9% mass production method of wasabi. The method of claim 1, Said cytokinin is a mass production method of horseradish, which is at least one substance selected from the group consisting of benzylaminopurine (BA), thiadiazuron (TDZ), kinetin (Kinetin) and zeatin (Zeatin). The method of claim 6, A mass production method of wasabi, which uses 0.01-1 mg / L of benzylaminopurine alone as a cytokinin. The method of claim 1, 0.01 mg / L of cytokinin in the solid medium, 0.01-1.0 mg / L of indolebutyric acid (IBA) mass production method of wasabi. The method of claim 1, In the growth step, the mass production method of wasabi was incubated for 1-2 months under conditions of 7-9 hours at a temperature of 10-20 ℃. The method of claim 1, In the growth step of wasabi was a mass production method of wasabi for cultivating wasabi plants in a bioreactor or culture vessel. The method of claim 10, A method for mass production of wasabi by eb and flood or air-lift when cultivating wasabi plants in a bioreactor. The method of claim 10, A mass production method of wasabi that cultivates wasabi plants in water. The method of claim 1, In the growth step, the temperature of 10-20 ℃, in a dark state, mass production method of wasabi for culturing wasabi for 1-2 months. The method of claim 1, After the growth step, the mass production method of wasabi further comprising the step of purifying wasabi plant grown in the liquid medium in the air. The method of claim 14, The purifying step is a mass production method of wasabi made of a condition of the temperature of 10-20 ℃ in a mixture of vermiculite and perlite in a ratio of 7: 3. The method of claim 14, After the step of purifying, the mass production method of wasabi further comprising the step of cultivating the purified horseradish plant. The method of claim 16, The nutrient solution cultivation is a mass production method of horseradish using any medium selected from the group consisting of hydroponic cultivation, solid medium single (pearlite) and solid medium mixed (pearlite: pitmos = 7: 3). Wasabi produced by the method according to any one of claims 1 to 17. Inducing roots in a solid medium containing a carbon source, auxin and agar by cutting the apex of the aseptic tissue cultured horseradish plant; And A method of mass-producing horseradish roots comprising the step of propagating the roots of horseradish roots derived from the solid medium in a liquid medium containing a carbon source and auxin. The method of claim 19, The solid medium is WPM (Woody Plants Medium) as a medium, containing 2-9% sucrose as a carbon source, 0.01-1mg / L of indolebutyric acid as auxin, pH adjusted to 4.8-7 Mass production method of wasabi root. The method of claim 19, In the proliferation step, the mass production method of horseradish root incubated for 1-2 months, at a temperature of 10-20 ℃, 7-9 hours. The method of claim 19, Mass production method of wasabi root with a root length of 1.2-1.7cm when inoculated with liquid medium in the growth step. The method of claim 19, In the growth step mass production method of wasabi root to cultivate wasabi root in the surface of the liquid medium in the bioreactor or culture vessel. The method of claim 19, The liquid medium is WPM (Woody Plants Medium) as a medium, containing 2-9% of sucrose as a carbon source, 3-5 mg / L of indolebutyric acid as auxin, pH adjusted to 4.8-7 Mass production method of wasabi root. The method of claim 23, wherein When cultivating wasabi root in the bioreactor, mass production method of wasabi root by the ebb and flood (ebb and flood) or air-lift (air-lift) method. The method of claim 19, The growth step is a mass production method of horseradish root incubated for 1-2 months in the temperature 10-20 ℃, dark. Horseradish root produced by the method according to any one of claims 19 to 26. Horseradish active ingredient extracted from horseradish or horseradish root mass-produced by the method according to any one of claims 1 to 26.

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