KR20110061073A - Method for mass production of taraxacum coreanum nakai by plant tissue culture - Google Patents

Abstract

PURPOSE: A method for producing a large amount of Taraxacum coreanum NAKAI by tissue culture technology is provided to be used in a food industry and medical industry. CONSTITUTION: A method for producing a large amount of Taraxacum coreanum NAKAI by tissue culture comprises: a step of collecting and cutting leaves of Taraxacum coreanum NAKAI young plants and placing the leaves on a MS solid medium containing 0.1-2 mg/L of auxin or 1-5 mg/L of cytokinine to induce callus or shoot; a step of culturing the induced shoot in a MS medium containing 0.1-1 mg/L of 2,4-D, 0.1-1 mg/L of NAA, 0.1-1 mg/L of IAA, or 0.1-1 mg/L of IBA to induce uprooting of the shoot; and a step of dipping the shoot in huponex for 10-40 minutes; a step of transplanting the shoot to a pot and culturing.

Description

Method for mass production of taraxacum using tissue culture {Method for mass production of Taraxacum coreanum Nakai by plant tissue culture}

The present invention relates to a method for mass-producing white dandelion using tissue culture technology, and the white dandelion plant re-divided by the method.

Dandelion, which is distributed throughout the world, has been used as a medicinal plant for a long time . Taraxacum coreanum Nakai, in particular, is a native dandelion that grows on both sides of low areas in Korea. These white dandelions are similar to yellow dandelions, but their flowers are white and are sometimes called 'Chosunpogong', 'Baekpogonggong' or 'Chosundang' (Kang et al., 2001). In oriental medicine, yellow dandelion ( T. mongolicum ), zombie ( T. hallaisanense ), mountain dandelion ( T. ohwianum ), western dandelion ( T. officinale ) and white dandelion ( T. coreanum ) This is called pogongyoung (浦 公 英), and is used as a laxative, tonic, and stomach medicine. In addition, dried roots more than 15 times above the ground is called pogongyeongeuneun (浦 公 英 根) and is used as an antipyretic, diuretic, expectorant, antidote, dandelion is different depending on the site.

In particular, the root contains many plant steroid compounds such as taraxasterol, beta-sitosterol, and sigmasterol, as well as chlorogenic, caffeic and ferrules. It contains phenolic compounds such as ferulic and chicoric acid (Akashi et al., 1994; Katrin et al., 2006; Wolbis et al., 1993). In addition, phenolic compounds of hydroxycinnamic acid are abundant in the above-ground part including leaves and flowers. Unlike the underground part, vitamin C and tocopherol, which are minerals such as potassium, calcium and iron, and antioxidants This is very high. In addition, dried leaves and stems were found to contain potassium in high amounts of 4.89% and 7.73%, respectively (Kang et al., 2000; Williams et al., 1996; Wilman and Derrick, 1994).

Therefore, dandelion has various physiological activities such as diuretics, anti-inflammatory, antioxidant, anti-cancer and anti-diabetic by such active ingredients, and is also known to be excellent in inhibiting the growth of enterobacteria as well as antibacterial activity.

Furthermore, dandelion has been used for food as well as for medicinal purposes. Young leaves are thin and soft, and have been edible for long time in China and Japan as ssammul and herbs, and in some regions, roots have been used as coffee substitutes.

Dandelion, however, can be said to be a useful plant that has been left unattended without fully utilizing the various functionalities and characteristics of each cultivar compared to the known efficacy. Moreover, the distribution of native white dandelions in our country is decreasing due to the high fertility and self-growth of western dandelions, and it is difficult to collect and harvest large quantities due to spring flowering and annual breeding. Therefore, in order to use white dandelion with excellent physiological activity as a resource, it is required to mass-produce through new technology to overcome the seasonal and geographical influence.

On the other hand, a plant is composed of a large number of cells to form a tissue, several tissues to form an organ, and various organs of different shapes and functions gather to form an organically controlled plant, the plant tissue culture is such a plant It is a technique of extracting and separating organs, tissues, and cells from cells and aseptically culturing them in a nutrient-containing medium on the plane to induce a population of callus or single cells or to regenerate them into fully functional plants. It is also called `` in-flight culture. ''

Materials used for tissue culture include various organs and tissues such as leaves, stems, roots, leaflets, flower beds, cotyledons and human scales of plants, and pears and endosperm, vesicles and protoplasts, which are part of seeds. Depending on the degree of differentiation of the plant material, the form can be generally classified into three types, namely, firstly, organizing plant material having a special organic structure such as pear or seed, and secondly, In some cases, de-differentiated and non-organized material is cultured like callus. Third, when cultivated organs or tissues separated into their intermediate forms, cells are de-differentiated to form callus, and the formed callus is re-differentiated to root. Sometimes they develop into organs such as stems or complete individuals.

In this way, the tissue culture of the plant may cause an irregular cell mass called a callus to be generated from tissues or cells extracted from the plant depending on the culture conditions, or regenerate new organs or plants.

Furthermore, the tissue culture technology of plants today reveals the physiological biochemical control mechanisms and gene expression mechanisms of plants, and is a good tool for mass production of useful materials using biotechnological techniques. Bulk breeding using cultures also produces disease-free, identical plants that are difficult to obtain by dispensing or cutting.

However, in the case of white dandelion, a native species of Korea, which has various useful physiologically active substances as described above, there has been no example of using tissue culture technology as a method for mass production.

Therefore, an object of the present invention is to provide a mass production method of white dandelion through tissue culture technology that can induce organ formation and shorten the regeneration time in the tissue of the plant to mass-produce white dandelion having various physiological activities will be.

In addition, another object of the present invention is to provide a white dandelion plant produced by the mass production method of the white dandelion according to the present invention.

In order to achieve the object of the present invention as described above, the present invention,

Harvesting and cutting leaves from seedlings of white dandelion, and inducing callus or shoots on a solid medium of MS containing 0.1-2 mg / L of auxin or 1-5 mg / L of cytokinin;

The induced differentiated shoots are at least one selected from the group consisting of 2,4-D 0.1-1 mg / L, NAA 0.1-1 mg / L, IAA 0.1-1 mg / L and IBA 0.1-1 mg / L Culturing on MS medium containing to induce rooting of shoots; And

When the induced rooting of the shoots grows to 5 cm or more, the shoots are immersed in 0.5-1.5% of Huponex for 10 to 40 minutes, then mixed with peatmoss, perlite and vermiculite. It provides a mass production method of white dandelion using tissue culture, comprising the step of culturing and growing by transplanting into a pot (pot).

In one embodiment of the present invention, the leaves collected from the seedlings of the white dandelion after surface sterilization of the seed of the white dandelion with 60 to 80% ethanol and 3 to 5% sodium hypochlorite (NaOCl), distilled water Washed with water, seeded and seeded in MS medium containing 2-4% sucrose and 0.2-0.5% gelite or 0.5-0.7% phytoagar, followed by germination. can do.

In one embodiment of the present invention, the leaves collected from the seedlings of the white dandelion may be the leaves of the white dandelion grown 7-10 weeks after germination.

In one embodiment of the present invention, the leaves collected from the seedlings of the white dandelion can be cut to a diameter of 5 ~ 10mm.

In one embodiment of the present invention, the peatmoss (peratmos), perlite (perlite) and vermiculite (vermiculite) may be mixed in a mixing ratio of 3: 1: 1, respectively.

In one embodiment of the present invention, the culture may be carried out under a temperature of 20 ~ 27 ℃, luminous intensity of 5000 ~ 15000 lux and photoperiod of 12 to 16 hours and dark cycle conditions of 8 to 12 hours. In addition, the present invention provides a plant of re-differentiated white dandelion produced by the mass production method of white dandelion using the tissue culture according to the present invention.

In the mass production method of the white dandelion according to the present invention, after extracting the leaves from the white dandelion, inoculated on a solid medium containing auxin or cytokinin to induce callus or shoots, and differentiated shoots contain a plant growth regulator Culturing on a medium to induce rooting of shoots, and then transplanting them into soil-filled pots to grow them, which is an optimal condition for mass production of white dandelions in a short time and at low cost. As a result, the white dandelion, which is a native of Korea, which contains a large amount of various pharmacological components, can be usefully used in various industries such as food industry and pharmaceutical industry.

The present invention is characterized in that it provides a method for mass production of white dandelion through tissue culture that can induce organ formation and shorten the regeneration time in the tissue of the plant for mass production of white dandelion.

In the mass production method of the white dandelion through the tissue culture according to the present invention, after cutting the leaves from the seedlings of the white dandelion, auxin 0.1 ~ 2mg / L or cytokinin 1 ~ 5mg / L containing MS Inducing callus or shoots on the solid medium of the callus; The induced differentiated shoots are MS containing one or more selected from the group consisting of 2,4-D 0.1-1 mg / L, NAA 0.1-1 mg / L, IAA 0.1-1 mg / L and IBA 0.1-1 mg / L Culturing on medium to induce rooting of shoots; And when the induced rooting of the shoots grows to 5 cm or more, the shoots are grown to 0.5-1.5% of hipponex (huponex). After soaking for 10 to 40 minutes, the method may include culturing and growing in a pot mixed with peatmoss, perlite and vermiculite.

More specifically, the method for mass production of white dandelion by tissue culture according to the present invention will be described for each step as follows.

Step 1: Callus  or Shoot  Induction stage

After disinfection of the seeds of the white dandelion, the seedlings are germinated by germinating them on a solid medium of the plant, and then tissues, preferably leaves, are cut from the germinated seedlings, and then cut on a solid medium to callus or shoots. Induce.

At this time, the seed of the dandelion can be sterilized by a method known in the art, preferably surface sterilized with 60 to 80% ethanol and 3 to 5% sodium hypochlorite (NaOCl).

The sterilized seeds were washed with distilled water, and then seeded in MS medium containing 2-4% sucrose and 0.2-0.5% gelite or 0.5-0.7% phytoagar. Germination can be induced, wherein the culture for induction of germination can be incubated under a temperature of 20 ~ 27 ℃, luminous intensity of 5000 ~ 15000 lux and 12 to 16 hours photoperiod and dark cycle conditions of 8 to 12 hours, Preferably it can be incubated in a culture room controlled to a temperature of 23 ~ 25 ℃ 10000 lux brightness and 16 hours photoperiod and 8 hours dark cycle conditions.

According to the above method, the seedlings of seedlings grown through subcultures at 2 week intervals may be cut to an appropriate size to induce germination from seeds, and then the size of the explants may be 5 to 10 mm. , Callus or shoots were wound on a solid medium of MS containing 0.1 to 2 mg / L of auxin 2,4-D (2,4-dichlorophenoxyacetic acid) or 1 to 5 mg / L of cytokinin BA (banzyladenine). Can be induced. Preferably, 5-7 mm fragments may be dented on a solid medium of MS containing cytokinin BA alone in an amount of 1.5-2.5 mg / L, to induce callus or shoots.

In the present invention, the solid medium of the MS can be used as long as it is a general MS solid medium used in the art for tissue culture, preferably 2-4% sucrose and 0.2-0.5% gelite or It may be a solid medium of pH 5.8 containing 0.5 ~ 0.7% phytoagar.

In addition, in the present invention, the leaves collected from the seedlings of the white dandelion may use the leaves of the white dandelion grown 7-10 weeks after germination, preferably the leaves grown 7 weeks.

On the other hand, it is important to note that in the first step, the regeneration rate of shoots may be greatly affected by the cutting timing and size of the leaf sections, so that the sections should be manufactured with the exact timing and size as possible.

Second step: Rooting  Induction stage

When the shoot is induced and elongated in the first step, the bottom of the shoot is cut off so that plant growth regulators are not added or the auxin hormone is transferred to the medium alone or mixed to induce rooting.

In this case, the shoots for inducing rooting may induce rooting by separating only the shoots of about 1.0 to 2.0cm, preferably about 1.5cm, from the leaf fragments of the differentiated shoots, and the isolated shoots are plant growth regulators. MS which is not added or contains one or more auxin hormones selected from the group consisting of 2,4-D 0.1-1 mg / L, NAA 0.1-1 mg / L, IAA 0.1-1 mg / L and IBA 0.1-1 mg / L It is possible to induce rooting by culturing on the medium, and preferably, NAA alone may be induced by incubating in a medium to which 0.4 to 0.6 mg / L, more preferably 0.5 mg / L is added.

In addition, the culture for induction of rooting is the same conditions as the culture for induction of the germination of the first step, that is, the temperature of 20 ~ 27 ℃, luminous intensity of 5000 ~ 15000 lux and photoperiod of 12 to 16 hours and 8 to 12 hours Can be incubated for 2-6 weeks under dark cycle conditions.

Step 3: Minutes ( pot Transplantation stage

When the rooting induction process in the cabin is completed by the second step, and the root growth of shoots is judged to be well performed, the shoots with root growth are removed from the cabin, and the existing medium components are completely removed, and then transplanted and grown in soil. Fully regenerated plants can be obtained.

The shoots for transplanting can be used to select the shoots with sufficient root growth from the tip of the shoots, preferably 5 cm or more root growth.

In addition, the shoots are washed with running water to completely remove the existing medium components, and then dissolved in water and used in chemical fertilizer components, preferably 0.5 to 1.5% of hippone (huponex) for 10 to 40 minutes, It can be grown in pots to produce fully shaped white dandelion plants.

At this time, the pot (pot) can be used to fill the soil for transplanting, the soil can be used both commercially available sterilized artificial soil or masato, preferably peatmos (peatmoss), perlite (perlite) ) And vermiculite may be mixed.

More preferably, the mixed soil may be a mixture of peatmoss, perlite and vermiculite in a mixing ratio of 3: 1: 1.

Therefore, the present invention can provide a mass production method of white dandelion through tissue culture through the above-described method, and can also provide a white dandelion plant produced and regenerated by the above method.

On the other hand, various plants, including dandelion, are known to show different differences in the regeneration conditions of shoots depending on the species. For example, the regeneration of plants from seven different varieties of leaf, stem and root tissues was investigated for strawberries. As a result, there have been reports of differences between varieties. In the case of tobacco, 58 organ redifferentiation experiments reported that 16 Australian native species and 28 South American native species showed higher differentiation rate under the same conditions than North American native species (Li. B et. al., Plant Cell Rep., 22, 4, 231-238,2003).

Therefore, the formation of organs from plant tissues shows different differences according to the varieties and native plants of each plant. Therefore, the dandelion, which is native to Korea, is unique to Korea and has excellent effects on the treatment and prevention of various diseases. It is very important to establish optimal regeneration conditions for white dandelions.

Therefore, the present invention optimizes the regeneration conditions of white dandelion with high efficiency through the process of forming the white dandelion directly from the tissue, and has a feature of providing a method for mass production of white dandelion through such conditions. .

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

White dandelion  Induction of germination from seeds

The white dandelion ( Taraxacum coreanum Nakai . ) Was used by the seed bank of Dongshin University, and the seeds were removed after tubular hair removal, surface sterilization for 3 minutes in 70% EtOH and 10 minutes in 4% NaOCL solution, and then sterilized. Washed with distilled water for 5 minutes. Thereafter, the sterilized seeds were sown in MS medium (3% sucrose, 0.4% gelite, pH 5.8) to induce germination. At this time, the culture was germinated by incubation with a 16 hour photoperiod and an 8 hour dark cycle of 10000 lux in a culture room controlled at 25 ± 2 ° C., followed by subculture at two week intervals.

< Example  2>

New  Judo

       Leaf shoot formation and callus formation rate of germ-free germ plants germinated in the aircraft of Example 1 was investigated. To this end, first, the leaves are collected from the germinated seedlings, and the leaves are cut to a size of 7 mm in diameter, and then BA, which is a type of 2,4-D or cytokinin, which is a type of auxin, is produced at each concentration. The treated MS medium (3% sucrose, 0.3% gelite, pH 5.8) was dentured and the concentrations of the treated 2,4-D and BA are shown in Table 1 below. After 4 weeks, the concentrations of auxin and cytokine affecting shoot formation and callus formation were investigated. In addition, the investigation of shoot formation according to the condition of the leaf tissue was tested using leaves 5, 6, 7, 8, 9 weeks after germination in the medium of BA 2.0 mg / L added, At this time, the culture conditions were performed under the same conditions as the culture conditions of Example 1.

TABLE 1

Concentrated 2,4-D and BA concentrations in mg / L

Treatment Material A B C D E F G H I 2,4-D 0 0.1 One 0 0.1 One 0 0.1 One BA 0 0 0 2 2 2 4 4 4

As a result, the fragments in the basic MS medium without the plant growth regulator did not show any change until 2 weeks after cultivation, but chlorophyll was destroyed over time and gradually changed along the cutting plane. (FIG. 1A). In the case of 0.1 mg / L of 2,4-D alone, callus was induced slowly around the cutout after 2 weeks of culture, but it did not show any further growth and gradually changed brown. 1b). On the other hand, no growth was observed in the medium condition with 1 mg / L of 2,4-D, but only gradually changed, and the tendency was more pronounced when mixed with BA. In the case of mixing 2 or 4 mg / L BA with 1 mg / L of 2,4-D, the browning reaction proceeded more rapidly as the concentration of BA increased, and tissue necrosis was observed in all sections at 3 weeks of culture. Could be done (FIGS. 1C, 1F and 1I). In addition, unlike the 2,4-D alone treatment of BA alone, no browning of the tissues was observed, and in the medium to which BA was added at 2 mg / L, callus formation did not occur, and nodule was formed immediately ( 1d and 3b), in the medium to which 4 mg / L of BA was added, the callus was induced to thicken the sections, and this phenomenon was also observed in the case of mixing 2,4-D and BA ( 1e, 1g and 1h).

In addition, dry weight and fresh weight of the leaves grown 5 weeks, 6 weeks, 7 weeks, 8 weeks, and 9 weeks after germination in the MS medium to which BA 2.0 mg / L was added were examined, as shown in FIG. 2. The dry weight and fresh weight were found to have the highest weight at 7 weeks.

< Example  3>

Malnutrition according to the leaves by growing season Formation rate  Research

Plant regeneration in the cabin has been known to show a large difference depending on the type of plant growth regulator and the physiological state of the plant. Therefore, the present invention after germination by the method of Example 1 to enhance the regeneration efficiency of the white dandelion according to the present invention by the growth period (leaves of 5 weeks, 6 weeks, 7 weeks, 8 weeks and 9 weeks) The leaves of white dandelion were harvested, and they were soaked in MS medium to which 2 mg / L BA was added, and then the rate of formation of maternal and malformed children was examined.

As a result, as shown in Figure 2, the white dandelion leaves at 7 weeks after germination, the highest rate of dendrite formation in the leaf sections was about 86%, compared to the leaves at other times, and the number of shoots generated per section Most frequently (see FIG. 3D). On the other hand, in the case of white dandelion leaves within 5 weeks, although about 58% of malaria was generated, it was difficult to secure a large number of explants due to the small leaf area, and the number of shoots generated was smaller than that of the 7th week (FIG. 3C). Reference). In addition, the rate of shoot formation per leaf section in the tissue at 8 weeks was about 45%, which was lower than that of 5-6 weeks, but the number of shoots was similar to 7 weeks. In the case of sections longer than 9 weeks in which no leaf area was increased in the cabin, the rate of formation of malformed buds was very low, and it was gradually changed from the edge of the section over time (see FIG. 2). .

Therefore, the above results showed that the formation of malaria from leaf explants was more efficient in immature tissues than in mature tissues. In mature leaf tissues, organ formation rate dropped sharply and too young leaf tissues had low shoot formation rate. In general, the leaves of the seventh week showed the best shoot and shoot germination.

< Example  4>

Rooting  Induction and minutes ( pot Transplant to

The shoots differentiated in Example 2 induced rooting by separating only the shoots of about 1.5 cm length from the leaf sections. To this end, rooting was induced by incubating the differentiated shoots for 2 weeks in a medium to which 2 mg / L BA was added. The rooting induction was based on a basal medium or a small amount of auxin (NAA), which was not added with plant growth regulators. , IBA, IAA and 2.4-D alone or 2,4-D alone were treated for each concentration, and after 4 weeks the rooting rate of each treatment group was investigated, and the results are shown in Table 2 below.

The transplantation process was taken from the tube with 5 cm or more root growth from the tip of the shoot, completely removed the medium, and then immersed in 1% Hyponex (USA) for 30 minutes, followed by peatmoss: perlite. ): Vermiculite was re-differentiated into complete plants by transplanting them respectively into mixed soil and masato mixed at a 3: 1: 1 mixing ratio.

TABLE 2

After 4 weeks of rooting induction, measurement results of rooting frequency according to plant growth regulators

Auxin (mg / L) Root formation frequency rate (%) Control group 13.3 ± 4.4 2.4-D 0.1 6.7 ± 3.3 2.4-D 0.5 0 ± 0 2.4-D 1.0 0 ± 0 IBA 0.1 16.7 ± 3.3 IBA 0.5 0 ± 0 IBA 1.0 20.0 ± 2.9 IAA 0.1 6.7 ± 4.4 IAA 0.5 38.3 ± 3.3 IAA 1.0 26.7 ± 3.3 NAA 0.1 15.0 ± 2.9 NAA 0.5 86.7 ± 4.4 NAA 1.0 43.3 ± 8.8

As a result, rooting took place after 3 weeks of culture, and rooting rate was found to be less than 5% in 1/2 MS medium without hormones (Result not shown). When treated, it was shown that rooting did not occur in the medium added in amounts of 0.5 mg / L and 1.0 mg / L, and callus was formed as the cut surface thickened. The same phenomenon was observed in the medium to which IBA was added at 0.5 mg / L. In addition, more than 50% shoots withered in the medium supplemented with IAA in an amount of 1 mg / L and died at the 4th week of culture, whereas the high rooting rate was over 85% in the medium added with 0.5 mg / L of NAA. (See FIG. 3E).

In addition, as a result of transplantation and re-differentiation into a complete plant, as well as mixed soil as well as Masato showed a high survival rate of 100% (see Fig. 3f), therefore, through these results, the inventors have found a white dandelion plant It can be seen that it can be directly implanted into the package without going through the purification step in artificial soil.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

1 is a photograph showing the callus and shoots formed from each leaf after cutting aseptically cultured leaves of white dandelion and wound on 2,4-D or BA alone or mixed with each concentration. A is a control group without any treatment, B is 0.1 mg / L only 2,4-D, C is treated with 1 mg / L only 2,4-D, D is 2 mg / L only BA, E is 2,4-D 0.1mg / L and BA 2mg / L, F is a mixture of 2,4-D 1mg / L and BA 2mg / L, G is 4mg / L only BA, H is 2,4- D 0.1 mg / L and BA 4 mg / L were mixed and treated, and I indicates that 2,4-D 1 mg / L and BA 4 mg / L were mixed.

Figure 2 is the number of shoots, shoot formation rate, dry age and fresh weight formed from leaves 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks after germination from seeds of white dandelion by the tissue culture technology of the present invention Is a graph comparing each.

Figure 3 is a photograph showing the plant shape of the dandelion re-divided into shoots, roots and minutes formed according to the method of the present invention, A is a shoot formed from the leaf tissue of white dandelion, B is added BA 2mg / L It shows shoots formed in the medium, C shows shoots formed from 5 weeks old leaves, D shows shoots formed from 7 weeks old leaves, and E shows roots grown for 4 weeks in medium added 0.5mg / ml NAA. F is the plant of the re-differentiated white dandelion grown by transplanting into a pot (pot) by the method of the present invention.

Claims (6)

Extracting and cutting leaves from the seedlings of the white dandelion, and inducing callus or shoots on a solid medium of MS containing 0.1-2 mg / L of auxin or 1-5 mg / L of cytokinin; The induced differentiated shoots are MS containing one or more selected from the group consisting of 2,4-D 0.1-1 mg / L, NAA 0.1-1 mg / L, IAA 0.1-1 mg / L and IBA 0.1-1 mg / L Culturing on medium to induce rooting of shoots; And When the induced rooting of the shoots grows to 5 cm or more, the shoots are immersed in 0.5-1.5% of Huponex for 10 to 40 minutes, and then, peatmoss, perlite and vermiculite are mixed. Method of mass production of white dandelion using tissue culture, comprising the step of culturing and growing by transplanting into a pot (pot). The method of claim 1, The leaves collected from the seedlings of the white dandelion are surface sterilized with 60 to 80% ethanol and 3 to 5% sodium hypochlorite (NaOCl) and then washed with distilled water and then 2 to 4% Using a tissue culture, characterized in that obtained by subculture after seeding and germinating in MS medium containing sucrose and 0.2 to 0.5% of gelite (gelite) or 0.5 to 0.7% phytoagar Mass production method of white dandelion. The method of claim 2, The leaves collected from the seedlings of the white dandelion is a mass production method of the white dandelion using tissue culture, characterized in that the leaves of the white dandelion grown in 7 to 10 weeks after germination. The method of claim 1, The leaves collected from the seedlings of the white dandelion is a mass production method of white dandelion using tissue culture, characterized in that the diameter is cut to 5 ~ 10mm. The method of claim 1, The peatmoss (peratmos), perlite (perlite) and vermiculite (vermiculite) is a mass production method of white dandelion using tissue culture, characterized in that each of the mixing ratio of 3: 1: 1. The method of claim 1, The culture is a mass production method of white dandelion using tissue culture, characterized in that carried out under a temperature of 20 ~ 27 ℃, luminous intensity of 5000 ~ 15000 lux and dark cycle conditions of 8 to 12 hours and photoperiod of 12 to 16 hours.

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