KR102609856B1 - Method for mass propagation culture of Trachomitum lancifolium(Russanov)Pobed - Google Patents

Abstract

The method for mass propagating and cultivating plants of the herbaceous herb of the present invention collects the stem internodes of the herbaceous herbaceae plants germinated from the seeds of the herbaceous herb, initializes them on a medium to obtain a culture, and performs each culture step of shoot induction, multiple shoot induction, and rooting induction. By using a suitable medium, we confirmed excellent results in growth characteristics such as survival rate, shoot development rate, shoot length, shoot number, rooting rate, root number, and root length of the herbaceous herb, and shoots, multiple shoots, and rooting were induced according to the above method. It can be usefully used to mass propagate fragrant herb with excellent survival rate and growth by planting the plant in the soil and acclimatizing it.

Description

Method for mass propagation culture of Trachomitum lancifolium (Russanov)Pobed}

The present invention relates to a method for mass propagating and cultivating plants of Gajeongyangpap using tissue culture, and specifically, to provide excellent growth characteristics in the steps of shoot induction, multi-truncation induction, and rooting from the stem internodes from which the seeds of Gajeongyangpap, an endangered species, have germinated. The present invention relates to a method for mass propagation and cultivation of fragrant herbaceae, which provides an optimal medium composition and exhibits excellent growth characteristics by acclimatizing seedlings in vitro.

Trachomitum lancifolium (Russanov) Pobed.) is a perennial plant of the Apocynaceae family. The size is about 40 to 80 cmn. The leaves are alternate on the main stem, and opposite on the branches, lanceolate or oval in shape, with extensions of leaf veins at the round ends. There are bumps. The flowers bloom in June and are purple and grow in perpendicular cone-shaped inflorescences, and the peduncles have fine hairs along with the calyxes. The fruit is bone-shaped, thin, and about 12cm long, and the seeds have hair-like terminal hairs.

Gajeongyangpul was discovered in Danyang, Chungcheongbuk-do by the Japanese Takenoshin Nakai on July 16, 1919, and existed only as a specimen (Seoul National University Herbarium). A photo of Gaejeongyangpul was taken in 1977 by Lee Young-no, director of the Korea Botanical Research Institute, before it bloomed. Although this is the last record, it was known to be extinct until it was rediscovered in Gyeonggi-do in 2005. As a result of the national red list evaluation, the fern is designated as a Critically Endangered plant. It is currently found in some areas along the west coast of Korea and in North Chungcheong Province, but its population is disappearing due to the adverse effects of the living environment. Although it is under threat, it is currently known to be distributed in small quantities in about 10 locations across the country.

As a salt-tolerant plant, it is a species that grows in soil with high salt concentration, desert areas, and riversides. It not only provides windbreaks and prevents sand loss, but is also an excellent fiber, medicinal, and tea raw material, and is said to improve ecological balance and soil. It has been reported and studied as a plant that can cope well with various abiotic stresses, including drought and salinity. In this context, Trachomium lancifolium (Russanov) Pobed.) is a plant that has strong germination ability and is resistant to cold, salt, and drought.

Salt-tolerant plants are plants that grow in areas where the salt concentration of the soil is high and general plants cannot adapt to. They are called salt-tolerant plants. They contain a lot of salt in their cells and have a high osmotic pressure, so they can absorb water even when the infiltration of soil solution is high. There is a peculiarity. There is no clear alternative for discovering replacement plants for landscapes in road construction sites with unfavorable plant growth conditions and poor soil conditions, and planted plants are dying due to the selection of plants unsuitable for the soil and environment, such as road cut sites and dry areas along riverbanks. Research on the selection of well-adapted and ornamental plant resources and methods for mass propagating them is urgently needed.

As a conventional prior art, Republic of Korea Patent Publication No. 10-2021-0036051 uses MS medium or 1/2MS medium as a basic medium and adds a growth regulator of BAP or IAA to induce shoots, seedling differentiation, and rooting from the internodal region of Salvia Salvia ginseng. And a method for mass propagation of Salvia Salvia is being disclosed by providing ex situ acclimated medium conditions. In addition, Republic of Korea Patent Publication No. 10-2020-0055417 cultivates the internodes of the jujube tree variety 'Bojo' on DKW medium supplemented with BAP, etc. to induce adventitious sprouts, shoots, seedling differentiation, and rooting, and in vitro acclimatization medium conditions. A method for mass propagation of jujube trees is being disclosed.

The above prior literature discloses a mass propagation method using tissue culture using the internodes of the plant, but the native habitat of the endangered species, Gajeongsangjapul, is very limited and rare, and no research has been reported on methods for cultivating and mass propagating it. .

Accordingly, the present inventor has developed a method for mass propagation through tissue culture of the endangered species, the herbaceous herb. We have developed the necessary basic technology, confirmed that excellent growth characteristics are shown during tissue culture in each stage of the present invention, and found and grown the optimal environment and soil to increase the in vitro acclimatization rate. A method for mass propagation and cultivation of the endangered species, Gajeongniang. was completed.

The purpose of the present invention is to germinate the seeds of the endangered species, ginseng, and mass propagation and culture methods using primary culture through stem culture, shoot induction, multi-tissue induction, rooting induction, and in vitro acclimation technology, and a mass propagation and culture method of fennel, which is an endangered species. We would like to provide cultured plants of Gajeonhyangpul.

In order to achieve the above purpose,

This invention

1) Germinating the seeds of clove grass in MS medium or WPM medium;

2) collecting the stem internodes of the germinated herbacea, placing them on 1/2MS medium or 1/2WPM medium, and superculturing them to obtain a herbaceous plant culture;

3) planting the stems of the fennel root culture obtained in step 2) on WPM medium, MS medium, 1/2WPM medium, 1/2MS medium, or 1/2WPM + 1/2MS medium to induce shoots and grow;

4) Inducing multiple shoots by planting the grown shoots on WPM medium containing BA or 1/4 WPM medium containing BA;

5) The base of the polytense-induced modified fragrant grass was mixed with 3/4WPM+1/6 MS medium, 1/4WPM medium with IBA, 1/2WPM medium with IBA, WPM medium with IBA, or BA. Inducing rooting by planting on WPM medium; and

6) Planting the rooting-induced plant of the herbaceous herb in soil and acclimatizing it in vitro.

The method for mass propagating and cultivating plants of the herbaceous herb of the present invention collects the stem internodes of the herbaceous herbaceae plants germinated from the seeds of the herbaceous herb, initializes them on a medium to obtain a culture, and performs each culture step of shoot induction, multiple shoot induction, and rooting induction. By using a suitable medium, we confirmed excellent results in growth characteristics such as survival rate, shoot development rate, shoot length, shoot number, rooting rate, root number, and root length of the herbaceous herb, and shoots, multiple shoots, and rooting were induced according to the above method. It can be usefully used to mass propagate fragrant herb with excellent survival rate and growth by planting the plant in the soil and acclimatizing it.

Figure 1 is an image of the plant and flower of Gajeonghyangpul.
Figure 2 is an image showing the germination of (A) 3 days after the seedlings of the wild clove seeds, and (B) 2 weeks after the seedlings.
Figure 3 is a diagram showing the shoot occurrence rate (%) through stem primary culture.
Figure 4 is a diagram showing shoot length (cm) through stem primary culture.
Figure 5 is an image showing the primary culture of Gajeonhyangpul stem and the growth state 2 weeks after primary culture.
Figure 6 is a diagram showing the callus incidence rate (%) from leaf explants.
Figure 7 is a diagram showing callus size (cm) from leaf explants.
Figure 8 is an image showing callus culture using Gajeonhyangpul leaves.
Figure 9 is a diagram showing the shoot occurrence rate (%) through stem culture.
Figure 10 is a diagram showing the number of shoots (units) and shoot length (cm) through stem culture.
Figure 11 is an image showing shoot induction of Gajeonghyangpul.
Figure 12 is a diagram showing the incidence rate (%) of multiple transit cities by medium.
Figure 13 is a diagram showing the average shoot length (cm) and the average number of shoots (units) in multi-stage induction for each medium.
Figure 14 is an image showing the development of multiple shoots through stem culture.
Figure 15 is a diagram showing the rooting rate (%) by medium after 4 weeks of culture during rooting induction.
Figure 16 is a diagram showing the number of roots (units), root length (cm), and stem growth (cm) after 4 weeks of culture during rooting induction.
Figure 17 is a diagram showing the induced rooting of Gajeonhyangpul for each medium.
Figure 18 is a diagram showing the survival rate (%) for each subsoil when acclimatization of the herb rooted through tissue culture.
Figure 19 is a diagram showing the number of roots (units), root length (cm), and living body length (cm) for each medium when acclimatizing the rooting of the herbaceous herb rooting on each tissue culture medium.
Figure 20 is an image showing the appearance of acclimatized seedlings 4 weeks after acclimatization of Gaejeonhyangpul. (Left: Horticultural topsoil, Right: Mixed topsoil)
Figure 21 is an image showing the appearance of acclimatized seedlings by medium and topsoil 4 weeks after acclimatization of Gaejeonhyangpul.

Hereinafter, the present invention will be described in detail.

This invention

1) Germinating the seeds of clove grass in MS medium or WPM medium;

2) collecting the stem internodes of the germinated herbacea, placing them on 1/2MS medium or 1/2WPM medium, and superculturing them to obtain a herbaceous plant culture;

3) A step of inducing shoots and growing the stems of the fennel root culture obtained in step 2) on WPM medium, MS medium, 1/2WPM medium, 1/2MS medium, or 1/2WPM + 1/2MS medium;

4) Inducing multiple shoots by planting the grown shoots on WPM medium containing BA or 1/4 WPM medium containing BA;

5) The base of the polytense-induced modified fragrant grass was mixed with 3/4WPM+1/6 MS medium, 1/4WPM medium with IBA, 1/2WPM medium with IBA, WPM medium with IBA, or BA. Inducing rooting by planting on WPM medium; and

6) It provides a method for mass propagation of the herbaceous herb, including the step of planting the rooting-induced herbaceous plant in soil and acclimatizing it in vitro.

In oriental medicine, the entire herb is called Napoma and is used medicinally. It is known to have anti-hypertensive, tonic, and diuretic effects, preventing heart disease, high blood pressure, and nervous breakdown. It has been reported to treat hepatitis, abdominal inflammation, and nephritis edema. As this useful medicinal resource plant, Gajeongniang grass is designated and managed as a protected species by the Korea Forest Service, so in order to use it as a raw material for medicinal products, the development of mass propagation technology is important. However, the seed germination rate and rooting rate of cuttings of Gajeongniang grass are very low, so the present inventor We attempted to super-cultivate the stem internodes of germinated Amaranth fragrant plants and prepare a mass propagation method using tissue culture technology.

Meanwhile, live propagation using plant seeds has the advantage of being able to multiply plants while preserving genetic diversity. However, this is difficult to apply to mass propagation of seeds with low germination rates, and has limitations in propagating plants with small populations such as endangered species and rare species.

The plant body is composed of various types of organs, such as roots, stems, leaves, flowers, etc. All organs have the ability to differentiate, but since the differentiation ability varies depending on the type of organ, selection of an appropriate culture organ is important. Most of the organs are the jugular part. It is cultured using (apical part, shoot tip), axillary bud, and leaf segment (leaf segment). The inherent differentiation ability of plants is called totipotency, which refers to the ability to regenerate a complete plant from a single cell or part of plant tissue. Although all cells have a typical ability, the expression results are significantly different depending on the degree of differentiation of cell tissue, collection site, medium composition, culture environment, etc., so optimization of culture conditions according to the type of plant or variety is necessary. .

In particular, plant tissues or organs require various nutrients to grow and proliferate in a special environment called an in vitro culture vessel (a culture container such as a culture test tube or glass bottle). Depending on the type of plant requiring nutrients or the type of cultured tissue or organ, Since there are differences depending on the type, a medium that suits the characteristics of the plant or tissue to be cultured must be developed. Therefore, the optimal medium composition must be selected and cultured according to the type of plant and the purpose of cultivation. At this time, in addition to the basic medium, specific inorganic salts, carbon sources, other organic substances, vitamins, and plant growth regulators are added or the concentration and content of some ingredients are added. It is necessary to change and use .

Specific examples of the commercially available culture media 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 C modified orchid), M (Mitra replate/maintenance), NN (Nitsch and Nitsch), Anderson, BDS (modified B5 of Dunstan and Short), and SH (Schenk & Hildebrandt basal salt) media. .

The medium used in the present invention may be WPM (woody plant mediem) medium or MS (Murashige and Skoog basal) medium, but most preferably, WPM medium can be used as the basic medium.

The WPM medium is a medium developed to cultivate shoots of trees and shrubs, and is widely used in commercial in-flight propagation of ornamental shrubs and trees.

The MS medium is the most commonly used medium in both dicotyledonous and monocotyledonous plants.

Based on the basic medium, the optimal medium composition can be selected by changing the concentration and content of some components depending on the type of plant and the purpose of cultivation. At this time, the components whose addition and content are changed include, but are not limited to, specific inorganic salts, carbon sources, other organic substances, vitamins, and growth regulators.

When preparing and using the culture medium, the culture medium may contain at least one selected from the group consisting of carbon sources, macroelements, trace elements, vitamins, and amino acids. The carbon source may be any one or more selected from the group consisting of sucrose, glucose, maltose, lactose, and fructose, and may specifically be sucrose. The carbon source may be included at a concentration of 20 to 40 g/L, specifically 23 to 37 g/L, and more specifically 26 to 33 g/L.

The macroelement is selected from the group consisting of MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O and K ₂ SO ₄ in WPM medium. It may include one or more of the following. Specifically, in the WPM medium, the MgSO ₄ ·7H ₂ O may be included at a concentration of 340 to 400 mg/L, preferably 350 to 390 mg/L, and more preferably 360 to 380 mg/L. In addition, the CaCl ₂ ·2H ₂ O may be included at a concentration of 70 to 120 mg/L, preferably 80 to 110 mg/L, and more preferably 90 to 100 mg/L. Additionally, the NH ₄ NO ₃ may be included at a concentration of 370 to 430 mg/L, preferably 380 to 420 mg/L, and more preferably 390 to 410 mg/L. Additionally, the KH₂PO₄ may be included at a concentration of 140 mg/L to 200 mg/L, preferably 150 mg/L to 190 mg/L, and more preferably 160 mg/L to 180 mg/L. In addition, the Ca(NO)₂·4H₂O may be included at a concentration of 530 to 580 mg/L, preferably 540 to 570 mg/L, and more preferably 550 to 560 mg/L. Additionally, the K₂SO₄ may be included at a concentration of 960 to 1,020 mg/L, preferably 970 to 1,010 mg/L, and more preferably 980 to 1,000 mg/L.

The macro element is any one or more selected from the group consisting of MgSO ₄ ·7H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, KCl, KI and KNO ₃ in MS medium. It can be included. Specifically, in the MS medium, MgSO ₄ ·7H ₂ O may be included at a concentration of 10 to 60 mg/L, preferably 20 to 50 mg/L, and more preferably 30 to 40 mg/L. Additionally, the NH ₄ NO ₃ may be included at a concentration of 970 to 1,030 mg/L, preferably 980 to 1,020 mg/L, and more preferably 990 to 1,010 mg/L. Additionally, the KH ₂ PO ₄ may be included at a concentration of 270 to 330 mg/L, preferably 280 to 320 mg/L, and more preferably 290 to 310 mg/L. In addition, the Ca(NO) ₂ ·4H ₂ O may be included at a concentration of 320 to 370 mg/L, preferably 330 to 360 mg/L, and more preferably 340 to 350 mg/L. Additionally, the KCl may be included at a concentration of 10 to 60 mg/L, preferably 20 to 50 mg/L, and more preferably 30 to 40 mg/L. Additionally, the KI may be included at a concentration of 40 to 90 mg/L, preferably 50 to 80 mg/L, and more preferably 60 to 70 mg/L. Additionally, the KNO ₃ may be included at a concentration of 970 to 1,030 mg/L, preferably 980 to 1,020 mg/L, and more preferably 990 to 1,010 mg/L.

The trace elements are FeSO ₄ ·7H ₂ O, MnSO ₄ ·H ₂ O, ZnSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, H ₃ BO ₃ , Na ₂ -EDTA and Na ₂ MoO ₄ · in WPM medium. It may include any one or more selected from the group consisting of 2H ₂ O. Specifically, in the WPM medium, the FeSO ₄ ·7H ₂ O may be included at a concentration of 5 to 50 mg/L, preferably 10 to 40 mg/L, and more preferably 20 to 30 mg/L. In addition, the MnSO ₄ ·H ₂ O may be included at a concentration of 10 to 35 mg/L, preferably 17 to 28 mg/L, and more preferably 20 to 25 mg/L. Additionally, the ZnSO ₄ ·7H ₂ O may be included at a concentration of 2 to 14 mg/L, preferably 4 to 12 mg/L, and more preferably 6 to 10 mg/L. Additionally, the CuSO ₄ ·5H ₂ O may be included at a concentration of 0.05 to 0.45 mg/L, preferably 0.10 to 0.40 mg/L, and more preferably 0.20 to 0.30 mg/L. Additionally, the H ₃ BO ₃ may be included at a concentration of 3.5 to 8.5 mg/L, preferably 4.5 to 7.5 mg/L, and more preferably 5.5 to 6.5 mg/L. Additionally, the Na ₂ -EDTA may be included at a concentration of 10 to 60 mg/L, preferably 20 to 50 mg/L, and more preferably 30 to 40 mg/L. In addition, the Na ₂ MoO ₄ ·2H ₂ O may be included at a concentration of 0.05 to 0.45 mg/L, preferably 0.10 to 0.40 mg/L, and more preferably 0.20 to 0.30 mg/L.

The trace elements are FeSO ₄ ·7H ₂ O, MnSO ₄ ·H ₂ O, ZnSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, H ₃ BO ₃ , Na ₂ -EDTA, Na ₂ MoO ₄ · It may include any one or more selected from the group consisting of 2H ₂ O and CoCl₂·6H₂O. Specifically, in the MS medium, the FeSO ₄ ·7H ₂ O may be included at a concentration of 5 to 50 mg/L, preferably 10 to 40 mg/L, and more preferably 20 to 30 mg/L. In addition, the MnSO ₄ ·H ₂ O may be included at a concentration of 1 to 12 mg/L, preferably 3 to 9 mg/L, and more preferably 5 to 7 mg/L. Additionally, the ZnSO ₄ ·7H ₂ O may be included at a concentration of 1 to 7 mg/L, preferably 2 to 6 mg/L, and more preferably 3 to 5 mg/L. In addition, the CuSO ₄ ·5H ₂ O may be included at a concentration of 0.005 to 0.045 mg/L, preferably 0.010 to 0.040 mg/L, and more preferably 0.020 to 0.030 mg/L. Additionally, the H ₃ BO ₃ may be included at a concentration of 0.5 to 3.5 mg/L, preferably 1.0 to 3.0 mg/L, and more preferably 1.5 to 2.5 mg/L. Additionally, the Na ₂ -EDTA may be included at a concentration of 10 to 60 mg/L, preferably 20 to 50 mg/L, and more preferably 30 to 40 mg/L. In addition, the Na ₂ MoO ₄ ·2H ₂ O may be included at a concentration of 0.005 to 0.045 mg/L, preferably 0.010 to 0.040 mg/L, and more preferably 0.020 to 0.030 mg/L. In addition, the CoCl₂·6H₂O may be included at a concentration of 0.005 to 0.045 mg/L, preferably 0.010 to 0.040 mg/L, and more preferably 0.020 to 0.030 mg/L.

The vitamins and amino acids may include one or more selected from the group consisting of glycine, inositol, nicotinic acid, pyridoxine, and thiamine in the WPM medium. Specifically, vitamins and amino acids may be included in the WPM medium at a concentration of 0.5 to 3.5 mg/L, preferably 1.0 to 3.0 mg/L, and more preferably 1.5 to 2.5 mg/L. Additionally, the inostol may be included at a concentration of 70 to 130 mg/L, preferably 80 to 120 mg/L, and more preferably 90 to 110 mg/L. The nicotinic acid may be contained at a concentration of 0.2 to 0.8 mg/L, preferably 0.3 to 0.7 mg/L, more preferably 0.4 to 0.6 mg/L, and the pyridoxine may be contained at a concentration of 0.2 to 0.8 mg/L, preferably 0.3 to 0.3 mg/L. It may be contained at a concentration of 0.7 mg/L, more preferably 0.4 to 0.6 mg/L. Additionally, the thiamine may be included at a concentration of 0.4 to 1.6 mg/L, preferably 0.6 to 1.4 mg/L, and more preferably 0.8 to 1.2 mg/L.

The vitamins and amino acids may include any one or more selected from the group consisting of inositol, nicotinic acid, pyridoxine, and thiamine in the MS medium. Specifically, vitamins and amino acids may be included in the MS medium at a concentration of inositol of 0.4 to 1.6 mg/L, preferably 0.6 to 1.4 mg/L, and more preferably 0.8 to 1.2 mg/L. The nicotinic acid may be contained at a concentration of 0.2 to 0.8 mg/L, preferably 0.3 to 0.7 mg/L, more preferably 0.4 to 0.6 mg/L, and the pyridoxine may be contained at a concentration of 0.04 to 0.16 mg/L, preferably 0.06 to 0.06 mg/L. It may be contained at a concentration of 0.14 mg/L, more preferably 0.08 to 0.12 mg/L. Additionally, the thiamine may be included at a concentration of 0.4 to 1.6 mg/L, preferably 0.6 to 1.4 mg/L, and more preferably 0.8 to 1.2 mg/L.

The mixed medium may be adjusted to pH 5.7 to 7.3, specifically pH 6.0 to 7.0, and more specifically pH 6.2 to 6.8.

The culture medium may be prepared as a liquid medium or a solid medium depending on the purpose of the culture, and when prepared as a solid medium, components such as agar or gelite may be additionally added. Specifically, agar may be included in the WPM medium or MS medium at a concentration of 7,700 to 8,300 mg/L, preferably 7,800 to 8,200 mg/L, and more preferably 7,900 to 8,100 mg/L.

The medium in step 1) of germinating the seeds of the above can be MS medium or WPM medium, but WPM medium is preferable.

The medium in step 2) of first culturing by pulverizing the stem internodes of the germinated Chinese herb may be 1/2MS medium or 1/2WPM medium, but is preferably 1/2WPM medium.

The shoot induction medium in step 3) can be WPM medium, MS medium, 1/2WPM medium, 1/2MS medium or 1/2WPM + 1/2MS medium, but is preferably 1/2MS medium or 1/2WPM + It is a 1/2MS medium, and most preferably it is a 1/2WPM + 1/2MS medium.

In the multi-passage induction medium of step 4), 0.1 to 2.0 mg/L of benzyladenine (BA) can be added to WPM medium or 1/4 WPM medium, preferably 0.4 to 1.6 mg/L, and further. Preferably, 0.8 to 1.2 mg/L can be added.

The rooting inducing medium in step 5) is 3/4WPM+1/6 MS medium, 1/4WPM medium added with indole butyric acid (IBA), 1/2WPM medium added with IBA, and WPM added with IBA. medium or WPM medium to which benzyladenine (BA) was added, preferably 3/4WPM+1/6 MS medium, 1/4WPM medium to which indole butyric acid (IBA) was added, and 1/4 to which IBA was added. It is a 2WPM medium, and most preferably it is a 3/4WPM+1/6 MS medium.

In step 5), 0.1 to 2.0 mg/L of indole butyric acid (IBA) may be added to the medium, preferably 0.4 to 1.6 mg/L, and more preferably 0.8 to 1.2. mg/L can be added.

Additionally, in step 5), 0.1 to 2.0 mg/L of benzyladenine (BA) may be added to the medium, preferably 0.4 to 1.6 mg/L, and more preferably 0.8 to 1.2 mg/L. L can be added.

Plant growth regulators are a general term for plant hormones involved in plant growth, such as auxins, cytokinins, gibberellins, abscisic acid, etc. This is included. Even during plant tissue culture, the appropriate type and concentration of growth regulator must be added to the medium for the culture to grow and differentiate according to the purpose of the culture.

Auxin is an essential hormone mainly involved in cell division in the stems and roots of higher plants. Since its ability to directly control cell division is weaker than that of cytokinins, it is used in combination with cytokinins to induce active growth and proliferation of cultures. It is common, and can be used primarily to induce root formation during tissue culture. Types of auxins include naphthaleneacetic acid (NAA), indolacetic acid (IAA), indole butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid.

(2,4-dichlorophenoxyacetic acid, 2,4-D), etc., but is not limited thereto.

Cytokinins are derivatives of adenine, one of the purine bases that make up nucleic acids. They mainly stimulate and promote cell division, but vary depending on the type of plant and the state of cell differentiation. It works. Cytokinins that can be used include kinetin, zeatin, benzyladenine (BA), 6-benzylamino purine (6-BAP or BAP), and diphenyl urea. However, it is not limited to this. In particular, kinetin, one of the types of cytokinins used in the present invention, promotes DNA synthesis during cell division, and BAP also promotes cell division. In particular, kinetin, one of the cytokinins, plays a role in promoting DNA synthesis during cell division, and benzylaminopurine (BAP) also functions to promote cell division.

In addition to the cytokinin and auxin, vitamins required for plant tissue culture include vitamin B complex such as thiamine HCl, nicotinic acid, pyridoxine HCl, riboflavin, and myo-inositol. There is a , which promotes the growth of in-flight cultures. In particular, thiamine is involved in carbohydrate metabolism and amino acid biosynthesis, pyridoxine HCl promotes cell growth and prevents aging, and nicotinic acid Silver is effective in preventing brown changes in callus, so it is effective when added during long-term subculture.

Inositol, added to the medium, is a type of hexitol that has the effect of promoting cell division and growth, and has little toxicity even at relatively high concentrations. The appropriate concentration generally used is 100 mg/L, and Myo-Inositol is a vitamin B complex with the greatest biological activity among the nine three-dimensional forms of inositol. It is known to be an essential element for growth in some plants.

The soil in step 6) can be horticultural topsoil or mixed topsoil, but is preferably mixed topsoil.

The horticultural topsoil may contain 1 to 7% of zeolite, 4 to 10% of perlite, 3 to 9% of vermiculite, 60 to 76% of coco peat, 10 to 20% of peat moss, and 0.05 to 0.35% of fertilizer, preferably It may contain 2 to 6% zeolite, 5 to 9% perlite, 4 to 8% vermiculite, 63 to 73% coco peat, 12 to 18% peat moss, and 0.10 to 0.40% fertilizer.

The mixed medium may contain 1 to 6% diatomaceous earth, 8 to 16% zeolite, 1 to 7% perlite, 20 to 30% vermiculite, 30 to 50% coco peat, 10 to 25% peat moss, and 0.05 to 0.45% fertilizer. and preferably contains 2 to 5% diatomaceous earth, 10 to 14% zeolite, 2 to 5% perlite, 22 to 28% vermiculite, 35 to 45% coco peat, 13 to 20% peat moss, and 0.15 to 0.35% fertilizer. You can.

The process of physiologically adapting a cultured plant that has been induced to root in-flight to a new environment or climatic condition is called acclimation. It is carried out naturally or artificially and is subject to complex environmental factors (seasonal changes, etc.) and specific conditions. This occurs within a limited range to which plants can adapt, such as changes in environmental factors (light intensity, etc.). Because of the anatomical and physiological conditions of plants grown in the air, it is important for survival to maintain high relative humidity in the early stages of transplantation into an acclimatization box.

The present invention provides a herbaceous herb grown by the mass propagation method of the herbaceous herb of the present invention.

In addition, the present invention provides a survival rate of 85 to 100%, a survival rate of 85 to 100%, a root count of 1 to 8, a root length of 2 to 10 cm, and a living body length of 4 to 12 cm when acclimatized in vitro to the soil in step 6). We provide a herbaceous herb that exhibits excellent growth characteristics.

In a specific example of the present invention, the present inventor used MS medium and WPM medium as basic media to perform tissue culture proliferation tests of the herbaceous herb (Table 1), and the callus induction, multi-hardening, and rooting induction media were used with inorganic dyes. A separate medium with adjusted amounts was prepared and used (Table 2). The average germination rate of seeds grown on MS medium and WPM medium was 91.65% (Table 3), and the germination state of the seeds of fennelia was confirmed 3 days after planting on MS medium and WPM medium and 2 weeks after planting. (Figure 2A and Figure 2B). As a result of primary culture of the stem internodes of the germinated herbaceous plant, the 1/2MS medium showed a shoot development rate of 66.7% and a shoot length of 7.5 ± 2.2 mm, and the 1/2WPM medium showed a shoot development rate of 83.3% and a shoot length of 6.5 ± 3.4 mm. It was confirmed that 1/2WPM medium, which has a higher shoot development rate during primary culture, is a more suitable medium (Table 4, Figures 3, 4 and 5). Callus was induced on the leaves of the primary culture of Gajeonhyangpul, and on WPM medium treated with 2,4-D at concentrations of 0.5 mg/L and 1.0 mg/L, all callus was generated with a callus generation rate of 100%, 2,4 WPM medium treated with -D at concentrations of 0.1 mg/L and 0.5 mg/L showed a callus size of 0.5 cm or more, resulting in the largest callus induction (Table 5, Figures 6, 7, and 8). As a result of inducing shoots from super-cultured Gajeongniper stems, the occurrence rate was 100% on 1/2WPM+1/2MS medium, the number of shoots was 4.0, the shoot length was 4.58cm, and the occurrence rate was 91.67% on 1/2MS medium. showed excellent growth characteristics with a shoot length of 3.67 and 4.0 cm, and the above two media were confirmed as suitable media for shoot induction (Table 6, Figures 9, 10 and 11). The 1/4WPM+BA 1.0mg/L medium for multiflora induction in the shoots of the above-mentioned fragrant herb showed the best growth characteristics, with multiflora occurrence rate of 84%, height growth of 3.72±2.01cm, and multiflora number of 4.92±1.29, and WPM+ In the treatment group with 1.0 mg/L of BA added, the growth characteristics were excellent, with a multiflorum occurrence rate of 72%, elongation growth of 4.12±2.55cm, and multiflora number of 2.32±1.49 (Table 7, Figures 12, 13, and 14). Rooting was induced by cutting off the base of the multi-cultivated herbaceous plant, and the 3/4 WPM + 1/6 MS treatment group showed the best growth characteristics with a rooting rate of 93.3%, number of roots 3.20 ± 1.32, and root length of 3.27 ± 1.25 cm. The treatment group in which 0.5 mg/L of IBA was added to 1/4WPM medium showed the next best results with a rooting rate of 73.3%, number of roots 3.13 ± 2.33, and root length of 1.3 ± 0.94 cm (Table 8, Figures 15, 16, and 16). 17). The herbaceous plants rooted in each medium were acclimatized in horticultural medium and mixed medium, and the names of raw materials and mixing ratios of each medium were recorded (Table 9). When treated with horticultural medium, the number of acclimatized seedlings rooted in 3/4WPM+1/6MS medium was 4.3±1.4, root length 6.0±2.3cm, and living length 8.3±2.6cm, and when treated with mixed medium, the number of roots was 4.6±0.6. , the root length was 6.5 ± 1.1 cm, and the living length was 8.8 ± 1.3 cm, so it was more appropriate to use mixed media when acclimatizing tissue culture seedlings of Gajeongniper (Table 10, Figures 18 and 19). In addition, the appearance of the acclimatized seedlings was confirmed after acclimatizing the rooted herbaceous plant in horticultural topsoil and mixed topsoil for 4 weeks (Figures 20 and 21).

Hereinafter, the present invention will be described in detail through examples.

However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

<Example 1> Collection, washing, and disinfection of Seeds of Gajeongjangseul

In a specific example of the present invention, the present inventor collected seeds of Gajeonghyangpul and used seeds stored in a 4°C refrigerated facility. Seeds of Gajeongniang were collected from the native habitat of Gajeongniang in Garak-ri, Oksan-myeon, Heungdeok-gu, Cheongju. Seeds of the refrigerated fragrant herb were washed in running tap water for 10 minutes, disinfected with 70% ethanol for 20 seconds, and then washed with distilled water 5 times for primary disinfection. The first disinfected seeds were washed for 10 minutes by adding 2 to 3 drops of Tween 20 to a 2.0% sodium hypochlorite (NaCIO) solution in a clean bench, and then washed 5 times with sterilized water.

<Example 2> Preparation of basic medium for tissue culture of Gajeongsangpul

The medium used in the tissue culture proliferation experiment of the revised fragrant herb was MS (Murashige and Skoog, 1962) and WPM (Lloyd and McCown, 1981) powder medium as the basic medium (Table 1). For callus induction, multi-tissue, and root induction media, separate media with adjusted amounts of inorganic dyes were prepared and used (Table 2). After adjusting the pH of the medium to 6.5, 20 ml of the medium prepared from agar and Gerlite was dispensed into each culture bottle and sterilized in a high-pressure sterilizer at 121°C for 15 minutes before use. The culture conditions were Cultured at an illumination intensity of 2,000 lux for 16 hours and at 25 ± 1 °C.

Composition (mg/L) basic A
(WPM) default B
(MS) macroelement MgSO₄·7H₂O 370 35 CaCl₂·2H₂O 96 - NH₄NO₃ 400 1,000 KH₂PO₄ 170 300 Ca(NO)₂·4H₂O 556 347 K₂SO₄ 990 - KCl - 37.3 KI - 65 KNO₃ - 1,000 NaH₂PO₄·H₂O - - Trace elements FeSO₄·7H₂O 27.8 27.8 MnSO₄·H₂O 22.3 6.06 ZnSO₄·7H₂O 8.6 4.0 CuSO₄·5H₂O 0.25 0.025 H₃BO₃ 6.2 2 Na₂-EDTA 37.3 37.3 Na₂MoO₄·2H₂O 0.25 0.025 CoCl₂·6H₂O - 0.025 vitamins and amino acids Glycine 2.0 - Inositol 100 1.0 Nicotinic acid 0.5 0.1 Pyridoxine·HCl 0.5 0.1 Thiamine·HCl 1.0 1.0 sucrose 30,000 30,000 agar 8,000 8,000 Gelite - - 2.4-D - - pH 6.5 6.5

Composition (mg/L) Shoot induction
(1/2WPM+1/2MS) Multi-Gyeongyudo
(1/4WPM+BA1.0) Root induction
(3/4WPM+1/6 MS) macroelement MgSO₄·7H₂O 202.5 92.5 101.25 CaCl₂·2H₂O 48 24 24 NH₄NO₃ 700 100 350 KH₂PO₄ 235 42.5 117.5 Ca(NO)₂·4H₂O 451.5 139 225.75 K₂SO₄ 495 247.5 247.5 KCl 18.65 - 9.325 KI 32.5 - 16.25 KNO₃ 500 - 250 NaH₂PO₄·H₂O - - 0 Trace elements FeSO₄·7H₂O 27.8 6.95 13.9 MnSO₄·H₂O 14.18 5.575 7.09 ZnSO₄·7H₂O 6.3 2.15 3.15 CuSO₄·5H₂O 0.1375 0.0625 0.06875 H₃BO₃ 4.1 1.55 2.05 Na₂-EDTA 37.3 9.325 18.65 Na₂MoO₄·2H₂O 0.1375 0.0625 0.06875 CoCl₂·6H₂O 0.0125 - 0.00625 vitamins and amino acids Glycine 1.0 0.5 0.5 Inositol 50.5 25 25.25 Nicotinic acid 0.3 0.125 0.15 Pyridoxine·HCl 0.3 0.125 0.15 Thiamine·HCl 1.0 0.25 0.5 sucrose 30,000 30,000 30,000 agar 8,000 8,000 - Gelite - - 3,500 2.4-D - - - B.A. - 1.0 - pH 6.5 6.5 6.5

<Example 3> Seed germination test of Gajeongniang grass

Germination rate was examined after 2 weeks by planting 10 seeds each in Petri dishes 3 times, sterilized on MS medium and WPM medium without growth regulators.

In the seeds placed on MS medium, the seeds swelled and began to germinate after 3 days. When the seed germination rate was examined after 2 weeks, the germination rate was 90.0%. In the seeds placed on WPM medium, the seeds also swelled and germinated after 3 days. When the seed germination rate was examined 2 weeks after starting, the germination rate was 93.3.0%, and the average germination rate for both media was 91.65%, showing a high germination rate (Table 3). The germination of the Seeds of Aspergillus fragrantwort was confirmed 3 days after planting them on the two media and the germination of the Seeds of Persimmons 2 weeks after planting (Figures 2A and 2B).

Results from seed culture on MS medium and WPM medium treatment area Number of teeth per iteration Average number of germination (units) Average germination rate (%) MS Badge 10 9 90.0 WPM badge 10 9.3 93.3 average 10 9.2 91.65

<Example 4> Initial culture using stem internodes of the herbaceous plant germinated in the seed germination test

In the seed germination test of Example 3, the stem internodes of the fragrant fragrant grass germinated were collected in 1/2MS and 1/2WPM medium modified with inorganic salts and no growth regulator added, and seeded in three repetitions of four each. By culturing, a culture of Gajeonhyangpul was obtained. After 2 weeks, shoot development rate, number of shoots, and shoot length were investigated.

One week after tooth formation, both 1/2MS medium and 1/2WPM medium showed shoot development, with 83.3% shoots occurring in 1/2WPM medium, and 66.7% shoot development rate in 1/2MS medium. As a result of the examination 2 weeks after the rooting, the shoot growth status of the 1/2MS medium showed a shoot length of 7.5 ± 2.2mm, and the 1/2WPM medium showed a shoot length of 6.5 ± 3.4mm, showing that the shoot growth rate was higher in the in-flight culture of the herbaceous herb. It was found to be suitable to use 1/2WPM medium modified with inorganic salts (Table 4, Figures 3 and 4). The collected stem internodes of Gajeongniper were placed on 1/2MS and 1/2WPM media modified with inorganic salts, respectively, to confirm the appearance during primary culture and the growth state 2 weeks after primary culture (Figure 5).

Results of primary culture through stem culture plant species badge average
Chi Sangsu Number of shoots (units) incidence rate
(%) Shoot length
(cm) Gajeonghyangpul 1/2 WPM 4 3.3 83.3% 6.5±3.4 1/2 MS 4 2.7 66.7% 7.5±2.2

<Example 5> Induction of callus in leaves of Gajeongniper

The leaves of the successful primary culture of Example 4 were used as experimental materials to induce callus by placing them on callus induction medium. The callus induction medium was WPM medium containing 3% sucrose and 0.3% gelrite with 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 0, 0.1. , 0.5, 1.0, 2.0, and 3.0 mg/L were treated and 10 ml each was dispensed into Petri dishes, and callus was induced by pricking 5 leaves per Petri dish 3 times.

As a result, callus was not induced in media that was not treated with 2,4-D, and callus was generated in all media that were treated with 2,4-D. However, in most calluses, only non-embryogenic calli were induced, without somatic embryos being induced. On WPM medium treated with 2,4-D at a concentration of 0.5 mg/L and 1.0 mg/L, all callus was generated from the leaf explants, and the callus development rate was 100%, and the callus development rate was 100%. WPM medium treated with /L and 2.0 mg/L induced 93.3% of callus, and WPM medium treated with 3.0 mg/L of 2,4-D induced 86.7% of callus, resulting in 2,4-D. It showed a better callus generation rate than the medium not treated with D (Table 5 and Figure 6). When the size of callus was expressed in diameter (cm), the medium treated with 2,4-D showed a callus size in the range of 0.25 to 0.59 cm, but the medium without 2,4-D treatment showed a callus size of 0.12 cm. The callus size was the smallest (Figure 7). In addition, it was confirmed that the WPM medium treated with 2,4-D at concentrations of 0.1 mg/L and 0.5 mg/L had the largest callus size, exceeding 0.5 cm (Figure 7). It was confirmed that callus was cultured from leaf explants, and callus died in the 4th week in all media (Figure 8).

Callus culture results from leaf explants of Gajeonhyangpul growth regulator Average number of dimensions (units) Average number of occurrences (units) Occurrence rate (%) Callus size (cm) note WPM+2,4-D(0) 5 0.3 6.7 0.12±0.05 Non-germination
callus WPM+2,4-D(0.1) 5 4.7 93.3 0.59±0.26 WPM+2,4-D(0.5) 5 5.0 100 0.57±0.13 WPM+2,4-D(1.0) 5 5.0 100 0.31±0.25 WPM+2,4-D(2.0) 5 4.7 93.3 0.26±0.11 WPM+2,4-D(3.0) 5 4.3 86.7 0.25±0.12

<Example 6> Induction of shoots from the stems of the herbaceous herb

In order to identify a growth regulator suitable for efficient shoot induction from the ginseng stems first cultured in Example 4, the fennel stems were grown on WPM medium, MS medium, 1/2WPM medium, 1/2MS medium, and 1/2WPM + 1/2MS medium. After 3 repeated woundings of 4 each, the shoots were observed for 3 weeks, and the shoot occurrence rate and growth status (shoot length (cm) and shoot number (units)) were investigated according to the treatment group.

As a result of shoot induction, the 1/2WPM+1/2MS medium showed an average occurrence rate of 100%, the number of shoots was 4.0, and the shoot length was 4.58 cm. On the 1/2MS medium, the occurrence rate was 91.67%, the number of shoots was 3.67, and the shoot length was 4.58 cm. The length was 4.0 cm, and among the treatments, the two treatments showed the highest shoot development rate and the longest shoot length (Table 6, Figures 9, 10, and 11). WPM and MS media show a low shoot induction rate of 75% compared to media with different inorganic salt concentrations, so 1/2WPM + 1/2MS medium and 1/2MS medium are the shoot induction media for Gajeongniper. It was found to be the most suitable (Table 6 and Figure 9). It was confirmed that shoot induction was performed on each medium of the herbaceous herb (Figure 10).

Results of shoot induction from the stems of the herbaceous herb by culture medium division Repeat equivalent constant W.P.M. M.S. 1/2WPM 1/2MS 1/2WPM+1/2MS Shinchosu (dog) 4 3.00 3.00 3.00 3.67 4.00 Shoot length (cm) 4 3.00 3.67 3.33 4.00 4.58 Occurrence rate (%) 4 75.00 75.00 75.00 91.67 100.00

<Example 7> Induction of multi-cultivation of Gajeonghyangpul

To induce multi-tissue stems by cutting the shoots of the induced herbacea, WPM medium without growth regulators was used as a base medium as a control, and as a test plot, 1/4 WPM medium with inorganic salts reduced by 1/4 was used as a base medium for the cytokinin system. BA 0.5 and 1.0 mg/L were added to medium containing 0.5 and 1.0 mg/L of benzyladenine (BA) at different concentrations, respectively, and 0.5 and 1.0 mg/L of BA were added to WPM medium, respectively, and the shoots of the herbaceous herb were repeated 5 times for each treatment group, and were treated in the light field. Four weeks after being infected with the disease, shoot occurrence rate and growth status (shoot length and number of shoots) were investigated according to treatment group.

Five days after rooting the shoots of the herbaceous herb into the medium, the development of multiple shoots was observed in the treatment group to which 0.5 mg/L of benzyladenine (BA) and 1.0 mg/L of BA were added to 1/4WPM medium, and 4 weeks after rooting. In the growth investigation results, the 1/4WPM+BA 1.0mg/L medium with the inorganic salts reduced by 1/4 showed the best results with a multi-tissue occurrence rate of 84%, kidney growth of 3.72±2.01cm, and number of shoots of 4.92±1.29. The next treatment that showed good results was the treatment with 1.0 mg/L of WPM+BA, which showed a 72% shoot occurrence rate, 4.12±2.55 cm kidney growth, and 2.32±1.49 shoots. Although the number of shoots was low, kidney growth was excellent. were investigated (Table 7, Figures 12 and 13).

Among the treatments excluding the control, the treatment that showed the lowest results was the treatment in which the inorganic salts were reduced by 1/4 and 1/4 WPM+ BA 0.5 mg/L and WPM+ BA 0.5 mg/L were added, and both the polydactyly occurrence rate and kidney growth were found to be low. (Table 7, Figures 12 and 13).

The most suitable conditions for multi-stage incubation of the herbaceous herb were 1/4 WPM + 1.0 mg/L of BA added, and the growth regulator benzyladenine (BA) was added to the medium at concentrations of 0.5 and 1.0 mg/L, respectively. When doing so, the higher the concentration of BA, the better the shoot development rate and multiple shoot induction situation (shoot number, shoot length) tended to be excellent (Table 7, Figures 12 and 13). When inducing multiple shoots, the kidney growth amount (cm) for each medium was confirmed (Figure 14).

Results of polyphylla induction from shoots of Gajeongsangpap by culture medium division Damage per repetition
(dog) Da-kyung
number of occurrences incidence rate
(%) average length
(cm) Average number of stems
(dog) W.P.M. 5 1.6 32 1.17 ±1.69 1.69 ±1.45 1/4WPM+ BA (0.5mg/L) 5 3.0 60 2.32 ±2.30 2.76 ±2.13 1/4WPM+ BA (1.0mg/L) 5 4.2 84 3.72 ±2.01 4.92 ±1.29 WPM+BA (0.5mg/L) 5 3.4 68 2.96 ±2.28 2.80 ±2.24 WPM+BA (1.0mg/L) 5 3.8 72 4.12 ±2.55 2.32 ±1.49

<Example 8> Induction of rooting of fragrant herb

In order to induce rooting by cutting off the base of the multi-tele shoots of the herbaceous plant induced during multi-cultivation induction culture, WPM medium with no growth regulator added was used as a base medium as a control medium, and 1/2 WPM with inorganic salts adjusted as a test medium was used as a basic medium to induce rooting. /4 WPM, 3/4WPM+1/6 Growth regulators added to MS medium and inorganic salt modified medium include auxin-type indole butyric acid (IBA) 0.5 mg/L and benzyladenine (BA) 1.0. ㎎/L was added to create a medium, and then 5 to 6 cells per treatment group were treated three times. After observing for 4 weeks, the rooting rate and growth status (root length, number of roots, etc.) according to treatment were investigated and the results are as follows.

From 2 weeks after planting, root development could be observed in all treatments except WPM (0mg/L, control) medium, which did not add growth regulators. As a result of the investigation after 4 weeks, the treatment that showed the best root development was the 3/4WPM+1/6 MS treatment with modified inorganic salts, which had the best roots with 93.3% rooting rate, 3.20±1.32 roots, and 3.27±1.25cm in root length. The results were shown, and the treatment group in which 0.5 mg/L of IBA was added to 1/4 WPM medium showed excellent results with a rooting rate of 73.3%, number of roots 3.13 ± 2.33, and root length of 1.3 ± 0.94 cm (Table 8 , Figures 15 and 16). Among the treatments in which growth regulators were added to the basic medium, the treatment that showed the poorest results was the addition of 0.5 mg/L of indole butyric acid (IBA) to WPM medium and 1.0 mg/L of benzyladenine (BA) to WPM medium. One treatment was investigated (Table 8, Figures 15 and 16). In addition, the rooting induced state of each culture medium was confirmed (Figure 17).

Results of rooting induction from multi-stage parts of Gajeongseonjapul by culture medium By badge Repeat equivalent constant (number) rooting
(dog) Rooting rate (%) average
Number of roots (units) average roots
Length (cm) average stem
Growth (cm) W.P.M. 5~6 0.7 13.3 0.13±0.35 0.30±0.80 0.33±0.88 1/4WPM +IBA 0.5 mg/L 5~6 3.7 73.3 3.13±2.33 1.3±0.94 2.11±1.49 1/2WPM +IBA 0.5 mg/L 5~6 3.09 60.0 1.60±1.50 1.40±1.24 1.70±1.49 WPM+IBA 0.5 mg/L 5~6 2.3 46.7 1.80±2.14 0.69±0.80 1.90±1.43 3/4 WPM+1/6 MS 5~6 4.7 93.3 3.20±1.32 3.27±1.25 7.20±2.47 WPM+BA 1.0 mg/L 5~6 2.7 53.3 1.07±1.16 1.17±1.36 3.63±0.58

<Example 9> Purification of Gajeongniper plants

In order to adapt to the external environment and determine appropriate soil conditions for forage transplantation, the plants rooted in the field were placed in an acclimatization seedling box (39cm They were filled with horticultural topsoil (Hanareum No. 2) and mixed topsoil (Golden Nara, Seongto No. 2), transplanted, and then installed in a double house. The names of raw materials and mixing ratios of horticultural medium and mixed medium are shown in Table 9 below (Table 9). The temperature inside the double house was maintained at 25 ± 3℃, and the humidity was maintained by sealing it for 3 days to maintain humidity. After a week, the plastic was opened to allow air to pass through, and the soil was watered with spray every day to prevent it from drying out. After 4 weeks of conducting the test, the results are as follows.

Topsoil raw material name and mixing ratio Topsoil for horticulture Mixed soil (horticulture + golden land) Raw material name Mixing ratio (%) Raw material name Mixing ratio (%) zeolite 4 zeolite 12 perlite 7 diatomaceous earth 3 Vermiculite 6 perlite 3.5 Coco Peat 68 Vermiculite 25.35 peat moss 14.8 Coco Peat 39 Fertilizer 0.2 peat moss 16.9 Fertilizer 0.25

As a result of the 4-week acclimatization test, the average survival rate by culture medium in the horticultural topsoil (70% oily topsoil: 30% masato) treatment was 73.3%, while the average survival rate by culture medium in the mixed medium treatment was 81.75%. When plants rooted in 3/4WPM+1/6MS medium were transplanted into mixed media, a 100% survival rate was observed.

Additionally, when plants rooted in 3/4WPM+1/6MS medium were transplanted into horticultural medium, a survival rate of 93% was observed. It is believed that plants rooted in 3/4WPM+1/6MS medium are very good for acclimatization (Table 10, Figure 18).

Among the horticultural medium treatments, 3/4WPM+1/6MS medium showed the highest root count count of 4.3±1.4, root length was 6.0±2.3cm, and living length was 8.3 during acclimatization of tissue culture seedlings. ± 2.6 cm, and in the mixed medium treatment group, the number of roots was 4.6 ± 0.6, which was a high result, and the root length was 6.5 ± 1.1 cm, and the living length was 8.8 ± 1.3 cm, showing the best results, showing the best results. When acclimatizing tissue culture seedlings, it was thought that using mixed media would be most appropriate (Table 10, Figures 18 and 19). The acclimatized seedlings rooted in each rooting inducing medium were acclimatized in horticultural topsoil and mixed topsoil for 4 weeks, and then the appearance of the acclimatized seedlings was confirmed (Figures 20 and 21).

Results of acclimatization test of fennel root rooted through tissue culture By topsoil By culture medium survival rate
(%) Number of roots (units) Root length (cm) Body length (cm) average Deviation average Deviation average Deviation For gardening
topsoil 1/4WPM+ IBA (0.5mg/L) 53 3.2 ±3.4 2.3 ±2.4 5.1 ±3.7 1/2WPM+ IBA (0.5mg/L) 67 3.6 ±3.1 3.4 ±2.7 4.9 ±3.8 WPM+ IBA 0.5mg/L 80 3.5 ±2.2 3.8 ±2.4 5.6 ±3.2 3/4 WPM+1/6 MS 93 4.3 ±1.4 6.0 ±2.3 8.3 ±2.6 mix
topsoil 1/4WPM+ IBA (0.5mg/L) 67 5.1 ±2.1 3.9 ±1.6 5.9 ±2.2 1/2WPM+ IBA (0.5mg/L) 73 4.5 ±2.4 4.1 ±1.0 7.2 ±2.0 WPM+ IBA 0.5mg/L 87 4.2 ±1.8 4.3 ±1.3 7.2 ±1.8 3/4 WPM+1/6 MS 100 4.6 ±0.6 6.5 ±1.1 8.8 ±1.3

Claims (13)

1) Germinating the seeds of clove grass in MS medium or WPM medium;
2) collecting the stem internodes of the germinated herbacea, planting them on 1/2WPM medium and primary culturing them to obtain a herbaceous herb culture;
3) a step of inducing shoots and growing the stems of the ginseng herb culture obtained in step 2) on 1/2MS medium or 1/2WPM + 1/2MS medium;
4) Inducing multiple shoots by planting the grown shoots on 1/4 WPM medium containing benzyladenine (BA);
5) A step of inducing rooting by placing the base of the multi-tissue induced multi-tissue base on 3/4 WPM + 1/6 MS medium; and
6) A method for mass propagation of the herbaceous herb, characterized in that it includes the step of planting the rooting-induced herbaceous plant in soil and acclimatizing it in vitro. delete delete delete The method for mass propagation of the herbaceous herb according to claim 1, wherein the medium in step 4) is a medium prepared by adding 0.8 to 1.2 mg/L of BA. delete delete delete The method for mass propagation of fragrant herb according to claim 1, wherein the soil in step 6) is horticultural topsoil or mixed topsoil. The method of claim 9, wherein the horticultural topsoil contains 1 to 7% of zeolite, 4 to 10% of perlite, 3 to 9% of vermiculite, 60 to 76% of coco peat, 10 to 20% of peat moss, and 0.05 to 0.35% of fertilizer. A method for mass propagation of fragrant incense grass, characterized in that. The method of claim 9, wherein the mixed medium contains 1 to 6% diatomaceous earth, 8 to 16% zeolite, 1 to 7% perlite, 20 to 30% vermiculite, 30 to 50% coco peat, 10 to 25% peat moss, and 0.05 to 0.05% fertilizer. A method for mass propagation of fragrant incense grass, characterized in that it contains 0.45%. delete delete