KR101500072B1 - In Vitro Propagation Method of Jeffersonia dubia Benth et. Hook - Google Patents

Abstract

The present invention relates to an in-flight reproduction method of a grasshopper grass, which comprises a first step of separating a segment from a seedling of a sterile-treated grasshopper, a step of dispersing the segmented product into a naphthalene acetic acid , NAA) and Ndi medium supplemented with Thidiazuron (TDZ), and cultured in shoots.

Description

{In Vitro Propagation Method of Jeffersonia dubia Benth et. Hook}

The present invention relates to an in-flight breeding method of a grasshopper grass, and more particularly, to a method for breeding a grasshopper grass which is a rare ornamental plant by investigating a plant growth regulator for shoot regrowth and in-flight rooting.

It is a perennial plant of the Berberidaceae family. It is also known as Chrysanthemum or Chrysanthemum. The flower is reddish purple and blooms in April to May. It is a very valuable domestic plant. It has high added value because it is used as a medicinal plant used not only for the efficacy of chewing, detoxification, and structure but also for treating various diseases.

The grasshopper is native to Korea and Southeast Asia and is currently designated as an endangered plant. It is very important to protect habitat by preventing extinction and artificially propagating endangered indigenous plants. Tissue culture methods are used to sustain the preservation of species through artificial proliferation.

Since endangered plants are prohibited to collect, export and distribute indiscriminately, it was very difficult to receive the plant material itself. Especially, since the growth period of the top part was very short about 4 months, it was much difficult to secure the plant material.

The grasshopper is generally propagated by the division of seeds or rhizomes, the rate of nutrition reproduction is very slow, and seed germination in natural habitat is also very insufficient due to morphological dormancy. Therefore, efficient breeding methods are necessary to preserve rare ornamental plants. Recent in vitro culture has emerged as an alternative for the preservation of rare plants. Some reports are available on in-flight breeding of barnacles, but no studies have been done on in-flight breeding of barnacles.

Accordingly, it is an object of the present invention to solve the above-mentioned problems, and to provide an in-flight breeding method of an effective grasshopper by investigating a plant growth regulator for shoot regrowth and in-flight rooting.

In order to achieve the above-mentioned object, the present invention provides a method of propagating in-flight reproduction of a grasshopper, comprising: a first step of separating a segment from a digest of a tuna grass treated with sterilization; and a step of separating the segment from a naphthalene acetic acid , And a second step of culturing shoots on a N6 medium supplemented with Naphthalene acetic acid (NAA) and Thidiazuron (TDZ).

In the second step, the naphthalene acetic acid (NAA) is 0.1 mg / L and the thidiazuron (TDZ) is 0.25 to 2.0 mg / L.

Another embodiment of the in-flight reproduction method of the present invention comprises a first step of separating an explant from a cotyledon of a sterile-treated turtle grass and a second step of separating the separated explant from a culture medium containing a Thidiazuron (TDZ) N6 medium, and culturing the cells in shoots.

In the second step, the thidiazuron (TDZ) is characterized by being in the range of 0.25 to 1.00 mg / L.

And a third step of elongating the shoot in N6 medium containing 0.1 to 4.0 mg / L of gibberellic acid (GA ₃ ).

Further, the method further comprises a fourth step of breeding in a medium of AM, MS, N6 and WPM supplemented with 0.1 to 0.5 mg / L of naphthalene acetic acid (NAA) do.

The medium is characterized by being an N6 medium.

In the in-flight bunting method of the grasshopper according to the present invention, the following effects are obtained.

It can effectively reproduce the rare ornamental plants, which can not only inhibit the extinction of the grasses, but also contribute to the productivity improvement of the grasses.

FIG. 1 is a graph showing shoots (TDZ concentration: 0.5 mg / L, NAA concentration: 0.1 mg / L) cultured in N6 medium supplemented with TDG and naphthaleneacetic acid (NAA) L, A: 7 days, B: 14 days, C: 28 days, D: Shincho kidney).
FIG. 2 is a graph showing the growth of direct seeded shoots in N6 medium supplemented with TDZ (TDZ concentration: 0.25 mg / L, A: 2 weeks, B: 3 weeks) , C: 4 weeks, D: 5 weeks)
FIG. 3 is a graph showing the growth of shoots after 4 weeks in N6 medium supplemented with gibberellin (GA ₃ ) according to the present invention. (A: GA ₃ concentration is 1.0 mg / L and B: GA ₃ concentration 2.0 mg / L)
FIG. 4 is a diagram showing the rooting appearance (NAA concentration: 0.5 mg / L, A: 4 weeks, B: 6 weeks) showing a rooting appearance in a 1/2 times N6 medium supplemented with napthalene acetic acid (NAA)
FIG. 5 is a view showing a picnic grass according to the present invention refining outdoors; FIG.

Hereinafter, a method of breeding expectation according to the present invention will be described in detail.

According to the present invention, the method of reproductive propagation can be classified into a method of in-flight regeneration using a grasshopper Dongjia and a method of direct irregular plant regeneration using a grasshopper. First, Will be described.

A first step; Separate the sections from the sterile-treated tumbler pool

After sterilization for cultivation of Dongjia, the slices are removed and the slice is placed on the medium.

A second step; The step of incubation with shoot

The above-mentioned slices are plated on shoots in N6 medium supplemented with naphthalene acetic acid (NAA) and Thidiazuron (TDZ). At this time, the naphthalene acetic acid may be added in an amount of 0.1 mg / L, and the thidiazuron (TDZ) may be added in an amount of 0.25 to 2.0 mg / L.

Step 3: Stretching the shoot

The shoot is cultured in N6 medium supplemented with Gibberellic acid (GA ₃ ). At this time, the gibberellin (GA ₃ ) may be added at a concentration of 0.1 to 4.0 mg / L.

Step 4; Step of breeding shoots

When the shoot-derived fragment is elongated by about 2 to 3 cm, it can be cultured in any one of AM medium, MS medium, N6 medium and WPM medium. At this time, the shoot is rooted.

(NAA) may be added, and the naphthalene acetic acid (NAA) may be added in an amount of 0.1 to 0.5 mg / L.

Meanwhile, a method of direct irregular shoot regeneration using a grasshopper, which is another embodiment of the present invention, will be described.

A first step; Separate the piece from the cotyledon of the sterile-treated tall grass

After the pasteurized paste is sterilized for cultivation, the shell of the cotyledon is removed and the piece is sutured to the medium.

A second step; The step of incubation with shoot

The slice is plated on N6 medium supplemented with Thidiazuron (TDZ) and cultured in shoots. At this time, the thidiazuron (TDZ) may be added at a concentration of 0.25 to 1.00 mg / L.

Step 3: Stretching the shoot

The shoot is cultured in N6 medium supplemented with Gibberellic acid (GA ₃ ). At this time, the gibberellin (GA ₃ ) may be added at a concentration of 0.1 to 4.0 mg / L.

Step 4; Step of breeding shoots

When the shoot-derived fragment is elongated by about 2 to 3 cm, it can be cultured in any one of AM medium, MS medium, N6 medium and WPM medium. At this time, the shoot is rooted.

(NAA) may be added, and the naphthalene acetic acid (NAA) may be added in an amount of 0.1 to 0.5 mg / L.

Hereinafter, the in-flight breeding method of the above-mentioned grasshopper grass will be described in detail through experimental examples.

[Experimental Example 1] - Regeneration in a cabin by using a dumbbell

1. Experimental materials and sterilization method

The grasshopper is protected by endangered plants, so it is very difficult to obtain plant material. They were officially cultivated in the greenhouse of Gyeongsang National University by purchasing officially a grasshopper at Gyeong Cheon Botanical Garden, which is a habitat for conservation of endangered plants. In order to obtain aseptic plants, Dongjia japonica was collected and washed in tap water for 20 minutes, rinsed 2-3 times with sterilized water and immersed in 70% (v / v) ethanol (EtOH) for 2 minutes for aseptic culture And then washed three times with sterilized water. Then, one drop of Tween 20 was added to 1.5% sodium hypochlorite (NaOCl), and the mixture was immersed for 15 minutes, washed three times with sterilized water, and sterilized to peel off two layers of the husk. And then immersed in 70% (v / v) ethanol (EtOH) for 2 minutes and then washed three times with sterilized water. Then, a drop of Tween 20 was added to 1.5% sodium hypochlorite (NaOCl), and the mixture was immersed for 15 minutes, washed three times with sterilized water, and then the remaining 2 to 3 layers of the skin were removed and 2-isopentyladenine 2-isopentyl adnine, 2-iP), benzyladenine (BA), and thidiazuron (TDZ) were cultured in a medium containing various concentrations.

2. Culture medium

Sucrose 3% (w / v) was added to various media such as Anderson's medium (AM), Murashige and Skoog medium (MS), Chu's medium (N6), and Woody Plant medium (WPM) 5.70, agar 0.8% (w / v) was added and sterilized at 121 for 15 minutes. (TDZ) was added to the medium subjected to high pressure sterilization after filtering. Other plant growth regulators were added to the medium prior to pH control.

3. Culture environment

The incubation temperature was set at 25 占 폚 and incubated under the conditions of 45 占 퐉 mol.m ^-2占^{-1 -1} for a known period of 16 hours. Three-wavelength cool-white fluorescent lamps [Philips 40 W tubes] were used as light sources in the culture room.

4. Results

In order to regenerate shoots of 2-iP, BA, and TDZ in Anderson's medium (AM), Murashige and Skoog medium (MS), Chu's medium (N6) and Woody Plant medium (WPM) After treatment at concentrations of 0, 0.25, 0.50, 1.0 and 2.0 mg / L, Dongjia of sterilized pupae were treated. After 4 weeks, the incidence of shoots and number of shoots per slice were examined in each treatment medium (Table 1).

(Effects of Media and Cytokinin on Shoot Development Rate and Shoot Occurrence Rate from Dong Jia, Concentration (mg · L ^-1 ) Shoot rate (%) Number of shoots per fragment 2-iP BA TDZ 2-iP BA TDZ

AM 0.00 13.2 d ^z 13.2 e 13.2 d 1.0 a 1.0 b 1.0 c 0.25  15.7 cd 24.4 d 42.6 c 1.0 a 1.0 b 1.0 c 0.50 18.0 c 30.0 c 56.2 b 1.0 a 1.0 b  1.8 ab 1.00 40.2 b 47.7 b  60.3 ab 1.0 a 1.0 b 2.0 a 2.00 46.8 a 59.0 a 62.4 a 1.2 a 1.6 a 1.4 b

MS 0.00  9.8 d  9.8 c  9.8 e 1.0 a 1.0 a 1.0 c 0.25 10.7 d  14.4 bc 27.6 d 1.0 a 1.0 a 1.0 c 0.50 16.2 c 19.2 b 53.2 a 1.0 a 1.0 a 1.2 b 1.00 26.8 b 23.7 a 41.0 b 1.0 a 1.0 a 1.8 a 2.00 33.0 a 25.2 a 38.0 c 1.0 a 1.2 a  1.6 ab

N6 0.00 24.4 e 24.4 e 24.4 e 1.0 b 1.0 c 1.0 c 0.25 36.2 d 27.0 d 49.7 d 1.0 b 1.0 c 2.2 b 0.50 40.0 c 32.4 c 66.3 c 1.0 b 1.0 c 3.8 a 1.00 48.7 b 54.6 b 78.0 a 1.2 b 1.6 b 3.6 a 2.00 63.2 a 70.8 a 73.0 b 1.5 a 2.0 a 2.7 b

WPM 0.00 11.0 e 11.0 d 11.0 d 1.0 a 1.0 b 1.0 b 0.25 16.7 d 19.4 c 24.2 c 1.0 a 1.0 b 1.0 b 0.50 21.3 c 33.2 b 60.4 a 1.0 a 1.0 b 1.6 a 1.00 27.8 b 51.6 a 49.4 b 1.2 a  1.2 ab  1.3 ab 2.00 39.4 a 53.0 a  0.0 f 1.3 a 1.4 a 0.0 d

Means followed by same letters within a column are not significantly diffenrent (P≤0.05)

Shoot incidence rate and number of shoots per section were the highest in N6 medium and lowest in MS medium. In terms of plant growth regulators, TDZ, benzyladenine (BA), and 2-isopentyladenine (2-iP) were higher in the order of shoot growth rate and number of shoots per shoot .

However, in the treatment of TDZ (2.00 mg / L), shoot growth rate and number of shoots per cutting slice were decreased. This result appears to be due to the toxicity of TDZ. As a result, it was found that the most suitable medium for shoot regeneration was TDZ at 1.00 mg / L in N6 medium.

Experiments were carried out in order to proliferate the regenerated shoots using Dongjia intercalation material. The experimental medium was prepared by adding 0.1 mg / L of taphthalene acetic acid (NAA) to each medium at a concentration of 0.25, 0.50, 1.0 and 2.0 mg / L of TDZ in N6 medium.

After 4 weeks, the incidence of shoots and the number of shoots per cut slice were investigated. When the concentration of TDZ was 0.50, the highest shoot rate (96.2%) and the number of shoots generated per slice (7.3) were the highest and the tendency was decreased as the concentration was higher , Fig. 1). The reason is also because of the toxicity of TDZ.

(Effect of TDZ and NAA Concentrations on Shoot Occurrence Rate and Shoot Occurrence Rate from Frogshell Plants Cultured in N6 Medium) TDZ (mg / L) Shoot rate (%) Number of shoots per fragment 0.25 61.4 d ^z 3.9 c 0.50 96.2 a 7.3 a 1.00 90.0 b 5.7 b 2.00 88.7 c 2.6 d

Means followed by the same letters within a column are not significantly different (P≤0.05).

[Experimental Example 2] Direct regeneration of adulthood using cotyledon grass

1. Experimental materials and sterilization method

The grasshopper is protected by endangered plants, so it is very difficult to obtain plant material. The seeds of the grasshopper were collected from the whole plants grown under the conditions of heavy humidity in Hanyang Botanical Garden, Kyonggi Province. Seeds were removed from the capsules, dried at room temperature (25 ± 3 ° C) for 3 days and stored at 5 ° C for 2 weeks. To combat morphological dormancy, the seeds were soaked in 1000 mg / L gibberellin (GA ₃ ) at room temperature for 24 hours and then dried at room temperature. The seeds were maintained at a temperature of 15/6 占 폚 for 30 days in a dark incubation for 12 hours / 12 hours in the photoperiod, and then kept at 5 占 폚 for 90 days. The treated seeds were surface sterilized in 70% (v / v) ethanol (EtOH) for 1 min and then washed three times with sterile water. Then, 1-2 drops of Tween 20 were added to 1.5% (v / v) sodium hypochlorite (NaOCl), soaked for 15 minutes, then immersed in 0.01% (w / v) HgCl ₂ for 10 minutes, Washed three times. The zygotic embryos were harvested from the seeds and cultured in a medium containing 2-isopentyl adenine (2-iP), benzyladenine (BA), and thidiazuron (TDZ) at various concentrations.

2. Culture medium

2-iP, BA and TDZ were treated with 0, 0.25, 0.50, 1.0 or 2.0 mg / L in Murashige and Skoog medium (MS) and Chu's medium (N6) / v), pH was adjusted to 5.70, agar 0.8% (w / v) was added and sterilized at 121 ° C for 15 minutes. (TDZ) was added to the medium subjected to high pressure sterilization after filtering. Other plant growth regulators were added to the medium prior to pH control.

3. Culture environment

The incubation temperature was set at 25 ± 1 ° C and cultured under light conditions of 45 μmol · m ^-2 · s ^-l PPFD in a culture room with a relative humidity of 70-80% for 12 hours. As a light source, a 3-wavelength cool-white fluorescent lamp (model FL 40EX-W, Kobsan Osram Co., Ltd.) having a small calorific value was used.

4. Results

Sterilized seeds were sown in the cabin to obtain 100% sterile plants. After 5 weeks of incubation, the incidence rate of shoots and number of shoots per cutting slice were examined in each treatment medium.

As a result, only callus, not shoot, was formed when 0.5-2.0 mg / L of TDZ was added to MS medium. In the N6 medium, shoot growth rate (72.1%) and shoot number per shoot (10.5) were the highest when TDZ concentration was 0.25 mg / L and the concentration of TDZ increased .

(Influence of TDZ and badge on direct adventitial regeneration from cotyledon grasses) TDZ (mg / L) Shoot rate (%) Number of shoots per fragment MS N6 MS N6 0.00 - - - - 0.25 - 72.1 ± 3.8 a ^z - 10.5 ± 1.6 a 0.50 Callus 66.3 ± 2.6 b Callus 8.0 ± 1.0 b 1.00 Callus 60.0 + - 3.0 c Callus 5.6 ± 1.4 c 2.00 Callus Callus Callus Callus

^z Means ± SE within a column followed by the same letters are not significantly different (P≤0.05).

The development of adventitia was successful in N6 medium, which seems to be due to low nutrient and nitrogen content. In addition, the type of nutrients added to the basal medium differs in MS and N6 medium, but the decisive factor for this is the ratio of ammonium and nitrate ions. The ratios of nitrate nitrogen and ammonium nitrogen in the MS and N6 medium were 66:34 and 80:20, respectively. In conclusion, the best way to induce adventitia using cotyledon grasses was to treat TDZ at 0.25 mg / L in N6 medium.

[Experimental Example 3] Direct regeneration of adulthood using cotyledon grass

1. Materials and Methods

1. Culture medium

3% (w / v) of sucrose was added to Chu's medium (N6) supplemented with 0.0, 0.1, 0.5, 1.0, 2.0 or 4.0 mg / L of gibberellin (GA ₃ ) , Agar 0.8% (w / v), and sterilized at 121 ° C for 15 minutes.

2. Culture environment

The incubation temperature was set at 25 ± 1 ° C and incubated for 16 hours under light conditions of 45 μmol · m ^-2 · s ^-l PPFD. As a light source, a 3-wavelength cool-white fluorescent lamp [Philips 40 W tubes] having a small heating value was used.

3. Results

To investigate the effect of gibberellin (GA ₃ ) on the kidney height of regenerated glutinous rice flour, it was applied to N6 medium supplemented with 0.0, 0.1, 0.5, 1.0, 2.0 or 4.0 mg / L of gibberellin (GA ₃ ).

After 6 weeks of incubation, the multiple shoots generated from the fragment failed to elongate shoots in the mixed treatment of TDZ or TDZ and NAA. Plant height increased as GA ₃ concentration increased from 0.1 to 2.0 mg / L, and plant height decreased to 4.0 mg / L (Table 4).

(The effect of GA ₃ on shoot height in grassy grass) GA ₃ (mg / L) Length of shoot (cm) 0.0 2.6 d ^z 0.1  2.8 cd 0.5 3.4 c 1.0 4.0 b 2.0 5.3 a 4.0  3.6 bc

^z Means ± SE within a column followed by the same letters are not significantly different (P≤0.05).

Shinso-kidney was most effective when the concentration of gibberellin (GA ₃ ) was 2.0 mg / L (Fig. 3). The plant was grown in N6 medium supplemented with gibberellin (GA ₃ ) 2.8, 3.4, 4.0, 5.3, and 3.3, respectively, when added at concentrations of 0.0, 0.1, 0.5, 1.0, 2.0, or 4.0 mg / L.

[Experimental Example 4] - Effects of the auxin-onset grass root

1. Culture medium

(AM) supplemented with 0.0, 0.1, 0.5, 1.0, 5.0 and 10.0 mg / L of indole-3-acetic acid (IAA), indolebutyric acid (IBA) and naphthaleneacetic acid ), Sucrose 3% (w / v) was added, the pH was adjusted to 5.80, agar 0.8% (w / v) was added and sterilized at 121 ° C for 15 minutes .

2. Culture environment

The incubation temperature was set at 25 ± 1 ° C and incubated for 16 hours under light conditions of 45 μmol · m ^-2 · s ^-l PPFD. As a light source, a 3-wavelength cool-white fluorescent lamp [Philips 40 W tubes] having a small heating value was used.

3. Results

In order to investigate the effect of auxin on the rooting of cabbage plants, various concentrations of auxin were treated in 1/2 x N6 medium when the regenerated shoots were 2-3 cm (Table 5).

(Effect of auxin on the rooting of grasses) Conc.
(mg / L) Rooting rate (%) Number of roots per second Root (cm) IAA IBA NAA IAA IBA NAA IAA IBA NAA 0.0 - - - - - - - - - 0.1 - -  92.4 b - - 4.0 b - - 2.8 b 0.5 - - 100.0 a - - 5.8 a - - 6.3 a 1.0 - - Callus - - Callus - - Callus 5.0 38.4 b ^z 78.6 b Callus 2.3 b 3.7 b Callus 1.6 b 3.2 b Callus 10.0 52.2 a 83.0 a Callus 3.0 a 4.3 a Callus 4.0 a 4.8 a Callus

- no response.

^z Means followed by the same letters within a column are not significantly different ( P ≤0.05).

After 6 weeks of incubation, root emergence rates, number of roots per root and root length were investigated. No shoots were produced in the medium containing no auxin, and the roots were transferred to the rooting medium supplemented with auxin. Two weeks later, shoots were produced at the ends of shoots. The rooting rate varied depending on the concentration of various auxins. Low concentrations of indole-3-acetic acid (IAA) or indolebutyric acid (IBA) did not cause roots at high concentrations.

Approximately 83% of shoots produced roots when added indolebutyric acid (IBA) at a concentration of 10.0 mg / L in N6 medium. The rooting rate (100%), root number (5.8), and root length (6.3 cm) were the highest at naphthalene acetic acid (NAA) concentration of 0.5 mg / The higher the concentration, the less the number of roots generated per second and the callus was formed. Further, when naphthalene acetic acid (NAA) was added at a concentration of 0.1 mg / L When added at the concentration, many roots were obtained.

[Experimental Example 5] Influence of the culture medium on rooting of cabbage grass

1. Culture medium

The regenerated shoots were prepared by adding 1 and 1/2 dilution of naphthalene acetic acid (NAA) 0.5 mg / L to Anderson's medium (AM), Murashige and Skoog medium (MS), Chu's medium (N6) After adding sucrose 3% (w / v), pH was adjusted to 5.80, agar 0.8% (w / v) was added and sterilized at 121 ° C for 15 minutes.

2. Culture environment

The incubation temperature was set at 25 ± 1 ° C and incubated for 16 hours under light conditions of 45 μmol · m ^-2 · s ^-l PPFD. As a light source, a 3-wavelength cool-white fluorescent lamp [Philips 40 W tubes] having a small heating value was used.

3. Results

When the regenerated shoots reached 2 to 3 cm, 1 and 2-fold dilutions of naphthalene acetic acid (NAA) 0.5 mg / L in AM, MS, N6, and WPM medium And then treated. After 6 weeks of culture, rooting rate, number of roots per root and root length were examined in each treatment medium (Table 6).

(Effect of the Medium Concentration on the Rooting of Pomaceae) Culture
medium Rooting rate (%) Number of roots per second Root (cm) Full
strength Half
strength B Full
strength Half
strength Full
strength Half
strength AM 61.0 c ^z  87.2 c  2.6 ab  5.4 ab 1.8 c  4.8 bc N6 68.4 b 100.0 a 3.0 a 5.8 a 2.7 b 6.3 a MS 27.2 d  66.4 b 2.4 b 4.0 c  3.0 ab 3.9 c WPM 80.4 a 100.0 a 1.8 c 5.2 b 3.6 a 5.2 b

^z Means followed by the same letters within a column are not significantly different ( P ≤0.05).

The number of roots was determined by adding 0.5 mg / L of naphthalene acetic acid (NAA) to AM, MS, N6, and WPM medium And N6, AM, MS, and WPM, respectively.

The highest rooting rate (100%) was obtained when the shoots were cultured in 1/2 times N6 medium and WPM medium. After 6 weeks, the number of roots (5.8) and root length (6.3 cm) were highest in the N6 medium at 1/2 dilution. Therefore, a low concentration of plant growth regulator is needed for in-cabbage rooting.

[Experimental Example 6] -External exfoliation of grass roots grown in the cabin

1. Experimental material

In order to exterminate the plant, the roots of the regenerated plant were washed with sterile water to completely remove the agar. The plant was implanted with a tosyloid soil composed of coir, peat moss, perlite and vermiculite in a purifying box requiring high relative humidity. The nutrient solution was fed once every two days and kept in the greenhouse. After 4 weeks, the clarified plants were transferred to a pot filled with Tosil.

2. Results

Experiments were carried out to establish an exo - purification system of plant rooted in the cabin. After 4 weeks, the plants exhibited a survival rate of 86% and were successfully purified (Fig. 5). After 3 weeks, the plants emerged with new leaves and were transferred to a port filled with Tosilite and purified in the greenhouse. Reproductive plants grew well without any problems in morphological and growth characteristics.

It is to be understood that the invention is not limited to the disclosed embodiment, but is capable of many modifications and variations within the scope of the appended claims. It is self-evident.

Claims (7)

A first step of separating the piece from the dentin of the sterile-treated tincture;
And a second step of culturing the shoots in a N6 medium supplemented with 0.1 mg / L of naphthalene acetic acid and 0.25 to 2.0 mg / L of titanaurine, In-flight reproduction method. delete A first step of separating the piece from the cotyledon of the sterile-treated tasty grass; And
And a second step of culturing the shoots in an N6 culture medium supplemented with tidia rhodoma at 0.25 to 1.0 mg / L to cultivate shoots. delete The method according to claim 1 or 3,
And a third step of elongating shoots in N6 medium containing 0.1 to 4.0 mg / L of gibberellic acid (GA ₃ ). 6. The method of claim 5,
And a fourth step of breeding in a medium selected from the group consisting of AM medium supplemented with 0.1 to 0.5 mg / L of naphthalene acetic acid (NAA), MS medium, N6 medium and WPM medium In-flight breeding methods of peculiar swimming pools. The method according to claim 6,
Wherein the culture medium is N6 medium.

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