KR20210104980A - Medium composition for in vitro culture of selaginella tamariscina and in vitro culture method of selaginella tamariscina using thereof - Google Patents

Abstract

The present invention relates to a medium composition for in vitro culture of Selaginella tamariscina and a method for in vitro culture of Selaginella tamariscina using the medium composition. Specifically, the medium composition for in vitro culture of Selaginella tamariscina according to the present invention promotes sporophyte formation from fragments of Selaginella tamariscina and increases live weight to be usefully used for the mass propagation of Selaginella tamariscina.

Description

Medium composition for the in-flight culture of the soybean and the in-flight culture method of the in-flight culture using the medium composition TECHNICAL FIELD

The present invention relates to a medium composition for in-flight culturing of basil, and a method for in-flight culturing of bas-relief using the medium composition.

Plants belonging to the genus Selaginella are representative medicinal ferns, and plants belonging to this group include various types of functional substances such as alkaloids, phenols, and terpenoids. Specifically, ginggetin, a biflavone isolated from Selaginella moellendorffii , exhibits anticancer activity by inducing apoptosis of cancer cells, and Sellaginella willdenowii . And Selaginella delicatula ( Selaginella delicatula ) Biflavonoids isolated from (biflavonoid) inhibit the growth of tumor cells. In addition, functional ingredients and their activities contained in various plants of the genus Celazinella are known.

A plant belonging to the genus Celazinella, baxson is also well known by the medicinal name of Gwonbaek, and has been used in folk medicine since ancient times to treat hemostasis, blood circulation, menstrual irregularities, menstrual pain, bruises, eohyeol, and pain. Functional ingredients such as amentoflavone, apigenin, hinokiflavone, isocryptomerin, sumaflavone, robustaflavone, tannin, etc. It has been reported to be effective in treating cancers such as throat cancer, lung cancer, cervical cancer, mammary gland cancer, skin cancer, kidney cancer, stomach cancer, rectal cancer, and liver cancer.

As the various activities of rock son are known, their medical value is increasing, but a systematic propagation method has not been developed. Most of the rocks currently in circulation are indiscriminately overfished in their natural habitats, which is why there are concerns about damage to their native habitats and a decrease in their numbers. In the past, the cutting method using leaves has been known as the propagation method of basalthorn, but this has a problem in that it is difficult to propagate in large numbers because it has low propagation efficiency and is vulnerable to changes in the environment. Therefore, there is a need for a stable supply method in response to the increasing demand for rock rock, and accordingly, the development of an efficient method for propagation of rock rock is required.

On the other hand, real-life propagation using plant seeds has the advantage of being able to propagate plants while preserving genetic diversity. However, this is difficult to apply to the mass propagation of seeds with a low germination rate, and there is a limit to propagating plants with small numbers such as endangered species and rare species.

Accordingly, a plant culture method capable of massively proliferating plants by inducing callus or somatic embryos from plant cells, tissues or organs and then re-differentiating them into complete plants is a method that can produce plants with the same genetic trait in large quantities at the same time. It is popular because it can. However, since the conditions or culture method of the culture medium are different depending on the type of plant, there is a need to establish a medium and method suitable for the plant to be mass-produced. In this regard, Korean Patent Registration No. 10-1204896 discloses a tissue culture method of Smilax china L. chinensis, which is used as a raw material for pharmaceuticals, health foods, and functional cosmetics, in a short time and in large quantities.

Republic of Korea Patent Registration No. 10-1204896

It is an object of the present invention to provide a medium composition for in-flight culturing of basalt, and a method for in-flight culturing of basalt using the medium composition.

Another object of the present invention is to provide a plant grown by the above method and a method of propagating the plant using the plant.

In order to achieve the above object, the present invention, based on the total volume of the entire culture medium, 0.1 to 10% (w / v) concentration of sucrose and 0.01 to 5% (w / v) of agar containing agar ( Selaginella tamariscina ) Provides a medium composition for in-flight culture.

In addition, the present invention provides a method for in-flight culturing of basalt, comprising the step of denting the basalt slices in the medium composition according to the present invention.

In addition, the present invention provides a plant grown by the method of the present invention.

Furthermore, the present invention provides a method of propagation of basalt, comprising the step of acclimatizing the plant of the present invention.

The medium composition for in-flight incubation of basalts according to the present invention can be usefully used for mass propagation of basalts by promoting sporophyte formation and increasing live weight from the dented basalts.

1 is 1/16 (A), 1/8 (B), 1/4 (C), 1/2 (D), 1 (E) or 2 ( F) is a diagram showing the results of culturing rock-son sections.
Figure 2 is a diagram showing the results of culturing by varying the length of the rock-son section to 3 (A), 6 (B) or 12 (C) mm in one embodiment of the present invention.
Figure 3 shows the concentration of sucrose in one embodiment of the present invention 0% (w / v) (A), 1% (w / v) (B), 2% (w / v) (C), 3% (w/v)(D), 4%(w/v)(E), or 5%(w/v)(F) is a diagram showing the results of culturing.
Figure 4 is cultured by varying the concentration of agar in an embodiment of the present invention to 0.6% (w / v) (A), 0.8% (w / v) (B) or 1.0% (w / v) (C) It is a diagram showing a result.
5 is a diagram showing the results of culturing by varying the nitrogen concentration to 7.5 (A), 15 (B) or 30 (C) mM in an embodiment of the present invention.
_{6 is a ratio of NH 4} ⁺ and NO ₃ ^- in an embodiment of the present invention 3:0(A), 2:1(B), 1:1(C), 1:2(D) or 0: 3(E) is a diagram showing the result of culturing differently.
7 is a diagram showing the results of acclimatization of the in-flight sporophyte of the in-flight in an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention is based on the total volume of the entire culture medium sucrose at a concentration of 0.1 to 10% (w/v) and 0.01 to 5% (w/v) of agar containing agar at a concentration of Selaginella tamariscina In- flight culture A medium composition for use is provided.

As used herein, the term " bawison ( Selaginella tamariscina )" is a fern in the genus Basisson, meaning a plant also known by the herbal name of gwonbaek. Basson is known to exhibit pharmacological activities such as hemostasis, antitumor, hypoglycemia, immunomodulation, and antibacterial, so it can be used for the treatment of various diseases.

The culture medium may be artificially prepared and used, or commercially available ones may be used. Specifically, the culture medium may include any one or more selected from the group consisting of carbon sources, mass elements, trace elements, vitamins and amino acids. For example, the carbon source may be sucrose.

The bulk element may be any one or more selected from the group consisting _{of CaCl 2} , KH ₂ PO ₄ , KNO ₃ , MgSO ₄ , KH ₂ PO ₄ , Ca(NO) _{2 and KCl, and specifically, the bulk element is CaCl 2} , KH ₂ PO ₄ , KNO ₃ , MgSO ₄ and KH ₂ PO ₄ may be any one or more selected from the group consisting of. More specifically, the bulk element may be CaCl ₂ , KH ₂ PO ₄ , KNO ₃ , MgSO ₄ and KH ₂ PO ₄ .

The bulk element can be used in an appropriate amount by a person skilled in the art. For example, the CaCl ₂ may be included in an amount of 280 to 400, 300 to 370, or 310 to 350 mg/L, and KH ₂ PO ₄ is 100 to 220, 130 to 200, or 150 to 190 mg/L in an amount of may be included. In addition, the KNO ₃ may be included in an amount of 1,500 to 2,500, 1,600 to 2,300 or 1,700 to 2,100 mg / ℓ, the MgSO ₄ may be included in an amount of 120 to 240, 140 to 220 or 160 to 200 mg / ℓ And, the KH ₂ PO ₄ may be included in an amount of 1,000 to 2,200, 1,200 to 2,000, or 1,400 to 1,800 mg/L.

In addition, the trace elements are CoCl ₂ ·6H ₂ O, CuSO ₄ ·5H ₂ O, FeNaEDTA, H ₃ BO ₃ , KI, MnSO ₄ ·H ₂ O, Na ₂ MnO ₄ ·2H ₂ O, ZnSO ₄ ·7H ₂ O and FeSO ₄ ·7H ₂ O may be any one or more selected from the group consisting of. Specifically, the trace elements are CoCl ₂ ·6H ₂ O, CuSO ₄ ·5H ₂ O, FeNaEDTA, H ₃ BO ₃ , KI, MnSO ₄ ·H ₂ O, Na ₂ MnO ₄ ·2H ₂ O, and ZnSO ₄ ·7H ₂ O may be any one or more selected from the group consisting of, more specifically, the trace element is CoCl ₂ ·6H ₂ O, CuSO ₄ ·5H ₂ O, FeNaEDTA, H ₃ BO ₃ , KI, MnSO ₄ ·H ₂ O, Na ₂ MnO ₄ .2H ₂ O and ZnSO _{4 .7H 2} O.

The above trace element may also be used in an appropriate amount by a person skilled in the art. As an example, the CoCl ₂ ·6H ₂ O may be included in an amount of 0.001 to 0.05, 0.01 to 0.04, or 0.02 to 0.03 mg/L, and the CuSO ₄ ·5H ₂ O is 0.001 to 0.05, 0.01 to 0.04 or 0.02 to It may be included in an amount of 0.03 mg/L, and the FeNaEDTA may be included in an amount of 10 to 60, 20 to 50, or 30 to 40 mg/L. In addition, the H ₃ BO ₃ may be included in an amount of 1 to 10, 3 to 8, 5 to 6 mg/L, and the KI is included in an amount of 0.1 to 1.5, 0.4 to 1.2, or 0.7 to 1.0 mg/L. may be, and the MnSO ₄ ·H ₂ O may be included in an amount of 5 to 25, 8 to 21, or 13 to 18 mg/L. In addition, the Na ₂ MnO ₄ ·2H ₂ O may be included in an amount of 0.001 to 0.05, 0.01 to 0.04, or 0.02 to 0.03 mg/L, and the ZnSO ₄ ·7H ₂ O is 1 to 15, 3 to 12 or 6 It may be included in an amount of from 10 mg/L to 10 mg/L.

The vitamins and amino acids can be used in appropriate amounts by those skilled in the art. The vitamins and amino acids may be at least one selected from the group consisting of glycine, myo-inositol, nicotinic acid, pyridoxine HCl and thiamine HCl, and specifically, glycine, myo-inositol, nicotinic acid, pyridoxine HCl and thiamine HCl. For example, the glycine may be included in an amount of 0.5 to 4, 1.0 to 3.5, 1.5 to 3.0 mg/L, and the myo-inositol may be included in an amount of 30 to 200, 50 to 150, 80 to 120 mg/L. It may be, and the nicotinic acid may be included in an amount of 0.1 to 1.0, 0.2 to 0.8, or 0.4 to 0.6 mg/L. In addition, the pyridoxine HCl may be included in an amount of 0.1 to 1.0, 0.2 to 0.8, or 0.4 to 0.6 mg/L, and the thiamine HCl may be included in an amount of 0.01 to 0.5, 0.05 to 0.3 or 0.08 to 0.2 mg/L. have.

On the other hand, specific examples of the commercially available culture medium include MS (Murashige and Skoog basal), WPM (Woody plant medium), B5 (Gamborg B5), W (White), LMWP (Lloyd and McCown Woody Plant basal), VW ( Vacin and Went), Km (Knudson Cmodified orchid), M (Mitra replate/maintenance), NN (Nitsch and Nitsch), AS (Anderson Salt), BDS (modified B5 of Dunstan and Short) and SH (Schenk & Hildebrandt basal salt) ) badges, etc.

The culture medium may be a solid medium. In addition, the culture medium according to the present invention may further include a growth regulator. For example, the growth regulator may be a cytokinin or auxin system, specifically, zeatin, kinetin, IAA (3-indoleacetic acid), NAA (naphthylacetic acid), IBA (indolebutyric acid) , gibberellin, BAP (N6-benzylaminopurine) and TDZ (thidiazuron) may be any one or more selected from the group consisting of.

The culture medium may be titrated to an appropriate pH known in the art, and may be modified as necessary by those skilled in the art.

The medium composition according to the present invention may include sucrose at a concentration of 0.1 to 10% (w/v) and agar at a concentration of 0.01 to 5% (w/v). Specifically, the sucrose is 0.1 to 10% (w / v), 0.5 to 10% (w / v), 1 to 10% (w / v), 0.1 to 8% (w / v), 0.5 to 8 % (w/v), 1 to 8% (w/v), 0.1 to 6% (w/v), 0.5 to 6% (w/v), 1 to 6% (w/v), 0.1 to 4 % (w/v), 0.5 to 4% (w/v) or 1 to 4% (w/v) concentration may be included. On the other hand, the agar is 0.01 to 5% (w / v), 0.1 to 5% (w / v), 0.5 to 5% (w / v), 0.01 to 4% (w / v), 0.1 to 4% ( w/v), 0.5 to 4% (w/v), 0.01 to 3% (w/v), 0.1 to 3% (w/v), 0.5 to 3% (w/v), 0.01 to 2% ( w/v), 0.1 to 2% (w/v) or 0.5 to 2% (w/v) concentration.

The medium composition may further include nitrogen. Specifically, the nitrogen is 1 to 50 mM, 3 to 50 mM, 6 to 50 mM, 1 to 40 mM, 3 to 40 mM, 6 to 40 mM, 1 to 30 mM, 3 to 30 mM, 6 to 30 mM , 1 to 20 mM, 3 to 20 mM, or 6 to 20 mM. For example, the nitrogen may include any one or more selected from the group consisting _{of NH 4} ⁺ and NO ₃ ^-. Specifically, when the NH ₄ ⁺ and NO ₃ ^- are included as a mixture, NH ₄ ⁺ and NO ₃ ^- are 1:0.5 to 5, 1:1 to 5, 1:1.5 to 5, 1:0.5 to 4 1 :1 to 4 1:1.5 to 4, 1:0.5 to 3 1:1 to 3 1:1.5 to 3, 0.5 to 5:1, 1 to 5:1, 1.5 to 5:1, 0.5 to 4:1, It may be added in a mixing ratio of 1 to 4:1, 1.5 to 4:1, 0.5 to 3:1, 1 to 3:1, or 1.5 to 3:1.

In addition, the present invention provides a method for in-flight culturing of basalt, comprising the step of denting the basalt slices in the medium composition according to the present invention.

The medium composition used for the in-flight culturing method of rockfish according to the present invention may have the characteristics as described above. For example, the medium composition may include sucrose at a concentration of 0.1 to 10% (w/v) and agar at a concentration of 0.01 to 5% (w/v) based on the total volume of the entire culture medium. In addition, the medium composition may further include 1 to 50 mM nitrogen.

The rock hand fragment may be obtained by a method known in the art. Specifically, the basalt fragment may be obtained by in-flight culturing of basal sporophyte, and may be a shoot tip portion of the basalt sporophyte.

In the case of in-flight incubation of basalthorn by the method according to the present invention, the basalt segment can be cut to an appropriate length and dented. Specifically, the section is 1 to 20 mm, 1 to 17 mm, 1 to 15 mm, 2 to 20 mm, 2 to 17 mm, 2 to 15 mm, 3 to 20 mm, 3 to 17 mm or 3 to 15 mm It can be cut to the length of

In addition, the present invention provides a plant grown by the method of the present invention.

The plant body may be one derived from the growth of the rock fragment in the same manner as described above. By denting the basalt fragments in the culture medium, it is possible to obtain basalt plants. At this time, the medium composition used may have the characteristics as described above, and specifically, the medium composition contains sucrose at a concentration of 0.1 to 10% (w/v) and 0.01 to 5 based on the total volume of the entire culture medium. It may include agar of % (w/v) concentration. In addition, the medium composition may further include 1 to 50 mM nitrogen.

Furthermore, the present invention provides a method of propagation of basalt, comprising the step of acclimatizing the plant of the present invention.

The basalt plant can be obtained in the same way as described above. As an example, the plant may be one derived from the growth of the basalt segment, and the induction thereof may be performed by dentin the culture composition having the characteristics as described above.

The acclimatization may be carried out by any method known in the art, and the acclimatized plant can be obtained as a proliferated rhododendron. The acclimatization may be cultured in any one or more types of soil selected from the group consisting of horticultural soil, unsupervised soil, fertile soil, and basal soil. At this time, the top soil may be used alone or mixed, and when used in a mixture, the mixing ratio may be appropriately adjusted by a person skilled in the art.

Hereinafter, the present invention will be described in detail by the following examples, provided that the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Anything that has substantially the same configuration as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.

Example 1. Preparation of basal sporophyte

The spores of the rock hand ( Selaginella tamariscina ) for mass propagation were prepared as follows.

Specifically, the rock hand grown in the glass greenhouse of the College of Agriculture, Life and Environment, Chungbuk National University was used. The spore spores of the spores were collected by a conventional method, and the surface was washed with distilled water. The washed rock spores were sterilized for 1 minute using 70% ethanol to sterilize the surface, treated with 2% sodium hypochlorite solution for 15 minutes, and washed 5 times with sterile water. Residual disinfectant was removed. The sporophyte of the rock hand from which the disinfectant was removed was cut into 10 mm lengths and cultured using MS medium (Murashige and Skoog, 1962). After culturing, the redifferentiated spores were subcultured at intervals of 4 weeks and used for mass propagation.

Example 2. Confirmation of medium concentration for in-flight culture of basal sporophyte

Using the redifferentiated sporophyte, the concentration of the medium for in-flight culture of basalt was confirmed as follows.

First, the shoot tip portion of the redifferentiated sporophyte in Example 1 was cut to a length of 10 mm and prepared as a section. On the other hand, based on the MS medium of the composition as shown in Table 1 below as a culture medium, the concentration of all nutrients contained therein is 1/16, 1/8, 1/4, 1/2, 1 or 2 times. A conditioned medium was prepared.

Furtherance Content ((mg/L) bulk element CaCl ₂ 332.02 KH ₂ PO ₄ 170.00 KNO ₃ 1900.00 MgSO ₄ 180.54 NH ₄ NO ₃ 1650.00 trace element CoCl ₂ 6H ₂ O 0.025 CuSO ₄ ·5H ₂ O 0.025 FeNaEDTA 36.70 H ₃ BO ₃ 6.20 KI 0.83 MnSO ₄ H ₂ O 16.90 Na ₂ MoO ₄ ·2H ₂ O 0.25 ZnSO ₄ 7H ₂ O 8.60 etc glycine 2.00 myo-inositol 100.00 nicotinic acid 0.50 pyridoxine HCl 0.50 Thiamine HCl 0.10 Sum 4405.19

50 ml of the prepared medium was dispensed into a culture vessel (Cat NO.310120, SPL Life Sciences, Korea) having a size of 120 × 80 mm, and the prepared sections were placed 6 each. After dentition, after culturing for 12 weeks at a temperature of 25±1.0°C, ^{a light amount of 30±1.0 μmol·m -2} ·s ^-1 , and a photogeometry of 16/8 h, the number and live weight of the formed spores were checked. It is shown in Table 2 and FIG. 1 .

Medium concentration Number of sporozoites/intercept Live weight (mg)/section 1/16 MS medium 11.23±1.94 22.42±4.73 1/8 MS medium 23.53±4.18 43.52±8.44 1/4 MS medium 65.67±6.64 221.03±16.55 1/2 MS medium 32.72±0.63 139.72±16.27 1 MS medium 8.59±0.97 27.12±3.12 2 MS medium 0.00 0.86±0.01

As shown in Table 2 and FIG. 1 , new sporophytes were formed in all media except for the case where 2 MS media was used. In particular, when 1/4 MS medium was used, 65.67 spores were formed, and the live weight of 221.03 mg was the highest, so the medium was the most effective for mass propagation of sections. On the other hand, the dentate section in 1 MS medium formed sporophyte, but some browning was confirmed, and the dentate section in 2 MS medium died after browning. Therefore, in subsequent experiments, 1/4 MS medium was used.

Example 3. Confirmation of section length for in-flight culture of sporophytes

Using the redifferentiated sporophyte above, the length of the section for in-flight incubation of rockfish was confirmed as follows. The experiment was carried out in Example 1, except that the shoot tip part of the redifferentiated sporophyte was cut into 3, 6 or 12 mm lengths from the apex, prepared in sections, and cultured by dentition in 1/4 MS medium. 2 and cultured under the same conditions and methods. As a result of the culture, the viability, the number of formed spores and the live weight were confirmed and shown in Table 3 and FIG. 2 .

Section length (mm) Survival rate (%) Number of sporozoites/intercept Live weight (mg)/section 3 75.00±8.33 29.23±4.35 32.37±4.70 6 87.50±7.98 28.07±1.20 30.15±4.00 12 88.89±11.11 48.72±2.22 81.78±1.49

As shown in Table 3 and FIG. 2, sporophytes were formed in all dentate sections regardless of the length of the sections, and the survival rates were also similar. In particular, the largest number of sporophytes was formed when a 12 mm-long section was dented, and the live weight was the highest. Therefore, thereafter, experiments were performed using sections with a length of 12 mm.

Example 4. Confirmation of the concentration of sucrose for in-flight culture of sporophytes

The concentration of sucrose for the in-flight culture of basalthorn using the redifferentiated sporophytes was confirmed as follows. Experiments, except that the sucrose content of 0, 1, 2, 3, 4 or 5% (w / v) was added to 1/4 MS medium, except that the 12 mm long section was dented and cultured, It was carried out under the same conditions and method as in Example 2. As a result of the culture, the number and live weight of the formed spores were confirmed and shown in Table 4 and FIG. 3 .

Sucrose Concentration (%(w/v)) Number of sporozoites/intercept Live weight (mg)/section 0 19.40±0.74 23.75±1.74 One 23.52±5.63 25.66±3.63 2 25.49±6.29 34.65±7.98 3 57.56±3.95 120.81±1.79 4 27.06±8.23 43.59±11.64 5 15.75±6.61 26.51±10.80

As shown in Table 4 and FIG. 3, sporophytes were formed in all dentate sections regardless of the concentration of sucrose added to the medium. In particular, the largest number of spores was formed in the medium to which sucrose was added at a concentration of 3% (w/v), and 19.4 spores were also formed in the medium to which sucrose was not added. On the other hand, in the medium to which sucrose was added at a concentration of 4% (w/v) or more, some sections were browned, and the resulting sporophytes were also browned, resulting in poor growth. However, except for the case where sucrose was treated at a concentration of 3% (w/v), no significant difference was observed.

Example 5. Confirmation of concentration of agar for in-flight culture of sporophytes

The concentration of agar for in-flight culture of baekson using the redifferentiated spores was confirmed as follows. In the experiment, the content of agar was added to be 0.6, 0.8 or 1.0% (w/v), and a 12 mm-long section was dented in 1/4 MS medium containing 3% (w/v) sucrose. Except for culturing, the same conditions and methods as in Example 2 were performed. As a result of the culture, the number and live weight of the formed spores were confirmed and shown in Table 5 and FIG. 4 .

Agar concentration (% (w/v)) Number of sporozoites/intercept Live weight (mg)/section 0.6 65.58±1.88 59.90±5.56 0.8 30.79±5.33 33.52±6.00 1.0 10.33±1.04 7.01±1.64

As shown in Table 5 and FIG. 4, sporophytes were formed in all dentate sections regardless of the concentration of agar added to the medium. Among them, when agar at a concentration of 0.6% (w/v) was added, 65.58 spores were formed and the most effective, and when agar at a concentration of 1.0% (w/v) was added, 10.33 spores were formed, the lowest showed growth.

Example 6. Confirmation of nitrogen concentration for in-flight culture of sporophytes

Using the redifferentiated sporophyte above, the concentration of nitrogen for in-flight culture of basalt was confirmed as follows. In the experiment, the total nitrogen content was added to be 7.5, 15.0 or 30. mM, and 12 mm in 1/4 MS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar. It was carried out under the same conditions and methods as in Example 2, except that the length sections were dented and cultured. As a result of the culture, the number and live weight of the formed spores were confirmed and shown in Table 6 and FIG. 5 .

Total nitrogen concentration (mM) Number of sporozoites/intercept Live weight (mg)/section 7.5 33.49±5.14 51.78±9.80 15.0 50.67±2.05 103.13±3.22 30.0 10.51±2.40 20.59±5.13

As shown in Table 6 and FIG. 5, sporophytes were formed in all dentate sections regardless of the concentration of total nitrogen added to the medium. In particular, when nitrogen at a concentration of 15 mM was added, 50.67 spores were formed the most, and the live weight was the highest.

Example 7. NH for in-flight culture of basal sporophyte ₄ ⁺ and NO ₃ ^- rate check

_{The ratio of NH 4} ⁺ and NO ₃ ⁻ for the in-flight culture of baekson using the redifferentiated sporophyte was confirmed as follows. The experiment is carried out so that the concentration of total nitrogen is 15 mM, at which time _{the ratio of NH 4} ⁺ and NO ₃ ⁻ is added to be 3:0, 2:1, 1:1, 1:2 or 0:3, 3 % (w / v) sucrose and 0.8% (w / v) 1/4 MS medium containing agar, except that the 12 mm long section was dented and cultured under the same conditions and methods as in Example 2 was performed with As a result of the culture, the number and live weight of the formed spores were confirmed and shown in Table 7 and FIG. 6 .

Ratio of NH ₄ ⁺ and NO ₃ ^- Number of sporozoites/intercept Live weight (mg)/section 3:0 0.00±0.00 0.99±0.04 2:1 26.02±6.29 36.44±9.06 1:1 45.12±9.81 67.19±21.39 1:2 57.61±5.62 120.90±5.19 0:3 0.00±0.00 1.13±0.01

As shown in Table 7 and FIG. 6 , sporophytes were formed in all dentate sections in the medium containing both _{NH 4} ⁺ and NO ₃ ^{− regardless of the ratio.} However, _{in the medium containing only NH 4} ⁺ or NO ₃ ^- , the dentate section was necrotic and did not form sporophytes. In particular, _{57.61 and 45.12 spores were formed in a medium containing NH 4} ⁺ and NO ₃ ⁻ in a ratio of 1:1 or 1:2, respectively, and thus the best effect was obtained. On the other hand, _{the sections cultured in a medium containing NH 4} ⁺ and NO ₃ ⁻ in a ratio of 2:1 formed sporophytes, but were partially browned.

Experimental Example 1. Purification of cultured spores

_{NH 4} ⁺ and NO ₃ ^- at a total nitrogen concentration of 15 mM, which is the medium condition for in-flight culture established through the above experiment, in a ratio of 1:2, 3% (w/v) sucrose and 0.8% ( w/v) acclimatization experiments were performed in an outdoor environment using the sections obtained by culturing 12 mm long sections in 1/4 MS medium containing agar.

Specifically, for external acclimatization, horticultural top soil (Hanareum No.2, Shinsung Mineral Co. Ltd., Korea) was prepared by filling a plastic pot with a diameter of 10 cm (Bummong Co. LTD., Korea). On the other hand, the lid of the culture vessel was opened to let outside air in for 24 hours before transplanting the slices of the in-flight to the outside environment. After 24 hours, the sections were washed with running water and transplanted into soil. The plastic port grafted with the section was placed in a 503×335×195 mm plastic box (SPC532(DP-D), SH Plastic, Korea), covered with a glass plate, and acclimatized under a humidity of 85±5.0%. At this time, head correlation is performed once a day ^{, and after acclimatization for 12 weeks at a temperature of 25±1.0°C, a light quantity of 43±1.0 μmol·m -2} ·s ^-1 , and a photogeometry of 16/8 h, the greenhouse was moved to

As a result, as shown in FIG. 7 , the in-vitro-cultivated lobe slices were grown to form a plant, and the survival rate was also excellent. Therefore, it was confirmed from the above that the medium composition established in the present invention can be usefully used to proliferate in large quantities through in vitro culture.

Claims (9)

0.1 to 10%, based on the total volume of the entire culture medium (w / v) concentration of sucrose and 0.01 to 5% (w / v) in-flight culture medium composition of the rock hand (Selaginella tamariscina) containing agar at a concentration of .
The medium composition of claim 1, further comprising nitrogen at a concentration of 1 to 50 mM.
According to claim 2, wherein the nitrogen is NH ₄ ⁺ and NO ₃ ^- containing any one or more selected from the group consisting of, the medium composition for in-flight culture of basalt.
According to claim 1, wherein the culture medium comprises any one or more selected from the group consisting of carbon sources, mass elements, trace elements, vitamins and amino acids, the medium composition for in-flight culture of rock son.
A method of in-flight culturing of basalthorn, comprising the step of denting the basalt fragments in the medium composition according to claim 1 .
[Claim 6] The method according to claim 5, wherein the fragment is a shoot tip portion of a rock hand.
The method of claim 5, wherein the basalt segment is 1 to 20 mm in length.
A basalt plant cultured by the method of claim 5 .
A method of propagation of a rock hand comprising the step of acclimatizing the plant of claim 8.

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