KR101955009B1 - Medium composition for in vitro culture of the herbal plant of Pinellia ternata and mass production method of the herbal plant of Pinellia ternata using the same - Google Patents

Abstract

[0001] The present invention relates to a culture medium for culture of oviducts and a mass production method using the same, and more particularly, to a culture medium containing 10 kinds of mediums, The plant growth regulator was optimized for each stage of the cultivation, and the auxin plant growth regulator was selected to facilitate the multiplication of individual cultivars.
When the mass production method of the multiparticulates and the seedlings produced by using the composition for cultivating eggs in accordance with the present invention is used, it is possible to mass produce the seedlings by satisfactorily purifying the soil.

Description

[0001] The present invention relates to a medium composition for culturing ova, and a method for mass production of the same using a culture medium containing the same,

BACKGROUND OF THE INVENTION Field of the Invention [0001] The present invention relates to a culture medium for culture in oviducts and a mass production method using the same.

Pinellia ternata (Thunb.) Breit] is a perennial herbaceous plant belonging to the genus Neptune. It is one of the medicinal plants which has used tuber as medicine for some time. The half-summer herb is a tuber which completely removed the juniper of the Araceae plant in the Korean Pharmacopoeia, and the herbal name is Pinelliae Tuber. The nature of the herbal medicines is new (hot) and has a pungent, strong spicy taste that is not legalized and toxic. It is mainly used for the treatment of asthma, sputum, headache, vomiting, dizziness, sore throat and the like. In addition, the potential value is high because it has recently been proven in various aspects such as obesity and nerve cell death suppression.

Inhabited by warm climatic conditions, it grows wild throughout the country, but the use of herbicides has caused a sharp decline in population. In addition, most of the labor depends on imports because of the large labor force to be harvested. The flower is bloomed and the fruit is bloomed and the seed is run, but the seed germination rate is low, so it is difficult to breed the seed, and it mainly grows as a tuber or a jujube. However, the differentiation of tubers is slow and the germination rate of jujube is very low. Many methods of in vitro culture have been attempted and active research has been carried out. In vitro culture of 2,4-D, BAP, Agar and Gelrite were applied to the culture medium to prepare small-sized tubers. Bacteria were treated with 0.1 mg / L 2,4-D, 0.5 mg / L BAP and Gelrite 4% And suggests an efficient method for the purpose of the study by inducing the differentiation of the plant and tubers in the inflorescence, and comparing the growth of the solid medium and the liquid medium, in the tissues such as tubers, leaves, seeds and juveniles There is a bar. Studies on the mass production of the undercounter have been carried out in a large number of studies in the cabin environment, and it is necessary to study the productivity improvement such as the difference of the tuber growth according to the environmental conditions such as the soil, temperature and humidity and the growth period.

Tissue incubation seedlings cultivated in airplane are to be settled in the field after soil purification. In order to facilitate soil mobilization at the time of purification, appropriate soil conditions should be provided for each plant. Since the rate of survival of cultured seedlings produced under the same conditions varies depending on the conditions of purification, the physical and chemical properties of the soil suitable for growth are different for each plant, and it is necessary to search soil conditions suitable for growth even under the conditions.

As a technique for mass production of such a plant, a plant growth regulator, a medium and a solid support disclosed in Korean Journal of Medicinal Chemistry 15 (3): 210 ~ 214, 2007, There is a description of the effect on the production of tubers. However, the culture medium for incubation of the present invention of the present invention and the mass production method using the same are not disclosed.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a culture medium for culture of egg yolk and a mass production method therefor using the same, The process conditions were established, and the conditions for mass production under the seedling through the purification of the produced albaine were established to complete the present invention.

In order to achieve the above object, the present invention is _{CoCl 2 · 6H 2 O, CuSO} 4 · 5H 2 O, FeNaEDTA, H 3 BO 3, KI, MnSO 4 · H 2 O, Na 2 MoO 4 · 2H 2 O, ZnSO ₄ , 7H ₂ O, KH ₂ PO ₄ , KNO ₃ , MgSO ₄ , CaCl ₂ , NH ₄ NO ₃ , myo-Inositol and thiamine hydrochloride as active ingredients. In-vitro culture medium composition for in vitro culture.

The present invention also relates to a method for producing a microorganism, comprising the steps of: (1) producing a microorganism by inducing the formation of multi-tuberous tubers by adhering a submerged nutrient to a culture medium containing an in vitro culture medium composition for production of an egg; And

(2) a step of environmental adaptation in the mixed soil in which the produced egg halves are mixed with Coir, Peat moss, Vermiculite, Perlite and Zeolite To provide a mass production method for the gravestones.

The present invention also provides a seedling produced by a mass production method.

The present invention relates to a culture medium for culture of oviducts and a method for mass production of the oviducts using the same, and the use of the culture medium for culture of oviducts according to the present invention enables the formation of multi- , And the production of egg yolk by the mass production method according to the invention according to the present invention enables the mass production of the seedlings to be carried out with good purification of the soil.

Fig. 1 shows the result of confirming the formation of small tubers (production of egg yolk) according to media 1 to 10 for optimal cultivation under the present invention.
2 is a graph showing changes in the growth index by adding plant growth regulator IBA (0.0 to 20.0 mg / L) to medium 7 according to the present invention.
FIG. 3 shows changes in growth index by adding plant growth regulator IAA (0.0 to 20.0 mg / L) to medium 7 according to the present invention.
FIG. 4 is a graph showing the change in mean fresh weight of the medium 7 according to the present invention by week (0 to 6 weeks) by adding sucrose by concentration (3 to 6%).
FIG. 5 is a graph showing the degree of formation of small tubers by in vitro culture according to addition of IAA (mg / L), BA (mg / L) and 0-6% sucrose by concentration to medium 7 according to the present invention. 1 was cultured under conditions that nothing was added except for medium 7; 2 was cultured in medium 7 supplemented with 3% sucrose; 3 was obtained by culturing in medium 7 supplemented with 6% sucrose; 4 contained 6% sucrose in medium 7 and 1 mg / l of BA was added to the cells; 5 contained 6% sucrose in medium 7, 1 mg / l of BA and 0.5 mg / l of IAA; 6 contained 6% sucrose in medium 7, 1 mg / l of BA and 1.0 mg / l of IAA; 7 contained 6% sucrose in medium 7, 1 mg / l of BA and 5.0 mg / l of IAA; 8 contained 6% sucrose in medium 7, 1 mg / l of BA and 10 mg / l of IAA; 9 contained 6% sucrose in medium 7, 1 mg / l of BA and 20 mg / l of IAA.
FIG. 6 is a graph showing the average growth index according to the addition of 0 to 20 mg / l of IAA, 0 to 1.0 mg / l of BA and 0 to 6% of sucrose (S) to medium 7 according to the present invention.
FIG. 7 is a photograph showing the production of the egg yolk in the mixed soil according to the present invention for 4 weeks and 8 weeks using the in vitro culture composition for the production of egg yolk according to the present invention.
(B) is a photograph of Alban Hai (Bar: 1 cm) cultivated in the medium, (C) showing the length after 8 weeks and the growth of the small tuber in the soil, , 1 was grown in soil A, 2 was grown in soil B, and 3 was grown in soil C.
FIG. 8 is a photograph showing the soil uptake (4 weeks or 8 weeks) from the production (0 day) to the soil according to the present invention.
FIG. 9 is a photograph showing the number of small tubers, fresh weight and net weight (%) after 25 weeks of soil purity at 0 day from the production according to the conditions of the present invention. C1 is a medium consisting of medium 7; C2 is medium consisting of medium 7 and 3% sucrose; C3 is a medium consisting of medium 7 and 6% sucrose; C4 is a medium consisting of medium 7, 6% sucrose and 1.0 ppm BA; C5 is a medium consisting of medium 7, 6% sucrose, 1.0 ppm BA and 0.5 ppm IAA; C6 medium medium consisting of medium 7, 6% sucrose, 1.0 ppm BA and 1.0 ppm IAA; C7 is a medium consisting of medium 7, 6% sucrose, 1.0 ppm BA and 5 ppm IAA; C8 is a medium consisting of medium 7, 6% sucrose, 1.0 ppm BA and 10 ppm IAA; C9 is a medium consisting of medium 7, 6% sucrose, 1.0 ppm BA and 20 ppm IAA.

The present invention relates to a process for the production of a metal oxide nanoparticle comprising CoCl ₂ .6H ₂ O, CuSO ₄ .5H ₂ O, FeNaEDTA, H ₃ BO ₃ , KI, MnSO ₄ .H ₂ O, Na ₂ MoO ₄ .2H ₂ O, ZnSO ₄ .7H ₂ O, KH _In -vitro culture medium for the production of egg yolk containing, as an active ingredient, ₂ PO ₄ , KNO ₃ , MgSO ₄ , CaCl ₂ , NH ₄ NO ₃ , myo-Inositol and thiamine hydrochloride .

The auxin plant growth regulator may further comprise an indole-3-acetic acid (IAA), an indole-3-acetic acid (IBA) -butyric acid, IPA (beta -indole propionic acid) and NAA (1-naphthaleneacetic acid), more preferably 1.0 to 5.0 mg / l of indole-3-acetic acid acid or an indole-3-butyric acid (IBA) in an amount of 5.0 to 10.0 mg / l, and more preferably 1.0 to 5.0 mg / l of an indole-3-acetic acid (IAA) It does not. Preferably, the cytokinin is BA (benzyladenine), more preferably 1 mg / L of BA, the amount of sucrose to be added is 3 to 6%, more preferably 6% no.

The content of the effective ingredient of the culture medium composition is 0.0125 to 0.0375 mg / l of CoCl ₂ .6H ₂ O, 0.0125 to 0.0375 mg / l of CuSO ₄ .5H ₂ O, 18.35 to 73.4 mg / l of FeNaEDTA, 3.1 to 12.4 mg / L H ₃ BO ₃ , 0.415 to 1.66 mg / l KI, 8.45 to 33.8 mg / l MnSO ₄ .H ₂ O, 0.125 to 0.5 mg / l Na ₂ MoO ₄ .2H ₂ O, 4.3 to 17.2 ZnSO ₄ · in _{mg / ℓ 7H 2 O, 135} ~ 540mg / ℓ of KH ₂ PO _4, 900 ~ of 3600mg / ℓ of KNO _3, 87.895 ~ 351.58mg / ℓ of _{MgSO 4, 83 ~ 332mg / ℓ} CaCl 2, that the 200 ~ 800mg / of _{ℓ NH 4 nO 3, 50 ~} 200mg / ℓ of myoinositol (myo-inositol) and 0.2 thiamine hydrochloride (thiamine HCl) of 0.8mg / ℓ not necessarily desirable, but not limited thereto. In addition, the form of the medium may be a solid medium or a liquid medium, preferably a solid medium, but is not limited thereto.

The present invention also relates to a method for producing a microorganism, comprising the steps of: (1) producing a microorganism by inducing the formation of multi-tuberous tubers by adhering a submerged nutrient to a culture medium containing an in vitro culture medium composition for production of an egg; And

(2) a step of environmental adaptation in the mixed soil in which the produced egg halves are mixed with Coir, Peat moss, Vermiculite, Perlite and Zeolite To the mass production method of the gravestone.

The mixed soil may contain 48 to 60% by weight of Coir, 10 to 14% by weight of Peat moss, 1.5 to 2.5% by weight of Vermiculite, 24 to 30% by weight of Perlite, 4 to 6% by weight of zeolite, but the present invention is not limited thereto. The environmental adaptation conditions are a temperature of 22 ° C, a light intensity of 67.5 μmolm ^-2 S ^-1 and a 16/8 hour light period, It is preferable, but not limited to, to grow for 6 to 8 days.

Further, the present invention relates to a seedling produced by a mass production method.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Induction of in-flight aseptic culture

Based on the seed and plant tissues (leaf, stem, roots, rootstock, etc.) of the antiprotozoa obtained by screening technology of genus (morphology, genetic confirmation), it is possible to cultivate in vitro by sterilizing the sample. The wild plants harvested from major domestic mountains were identified by morphological classification and genetic testing, and then used as materials for in vitro culture.

Tuber, leaf, seed, juice, etc. were surface-sterilized with primary distilled water and 100% ethanol to induce in-flight aseptic plant, then immersed in 70% (v / v) ethanol for 30 seconds and washed with sterile water. Sodium hypochlorite solution was sterilized in 3% (v / v) sodium hypochlorite for 10 min. The washed samples were washed with 3% (w / v) sucrose and 0.45% (w / v) (MB Cell, USA) was used. The pH was adjusted to 5.6 ~ 5.8 and sterilized at 121 ℃ for 15 min. The culture was carried out in an incubator with an internal temperature of 25 ± 1 ° C and a luminous intensity of 5,000 lux for a 16/8 hour light-dark cycle. For the purpose of inducing plant and small tuberculosis, in vitro cultures were induced by subculture and subculture in a fresh medium of the same conditions until the desired time.

The plant growth regulators (auxin) and cytokinine regulators (plant growth regulators) were selected after selecting the tissues (tubers, leaves, seeds, juveniles) To induce plant regeneration or to induce dedifferentiation (callus organism).

Example  2. Optimal production of in vitro culture

In order to investigate the optimum medium composition for the production of multi - cultivated microbial populations optimized by in vitro culture, 10 cultivars (medium 1 ~ 10) were used. The medium used in the present invention was purchased from Duchefa and added with 3% (w / v) sucrose and 0.45% (w / v) agar (phyto agar).

Medium 1 used in Example 2 contained 1650 mg / l NH ₄ NO ₃ , 1900 mg / l KNO ₃ , 440 mg / l CaCl ₂揃 2H ₂ O, 370 mg / l MgSO ₄揃 7H ₂ O, 170 mg / l K ₂ PO ₄ , 0.83 mg / l KI, 6.2 mg / l H ₃ BO ₃ , 22.3 mg / l MnSO ₄ .4H ₂ O, 8.6 mg / l ZnSO ₄ .7H ₂ O, 0.25 mg / l of Na ₂ MoO ₄ .2H ₂ O, 0.02 mg / l CuSO ₄ .5H ₂ O, 0.025 mg / l CoCl ₂ .6H ₂ O, 43 mg / l Ferric-EDTA, 3% sucrose, , pH 5.7, 100 mg / l of inositol, 0.5 mg / l of nicotinic acid, 0.5 mg / l of pyridoxine.HCl, 0.4 mg / l of thiamin.HCl, A medium consisting of 30 mg / l of indoleacetic acid (IAA) and 0.04 to 10 mg / l of kinetin,

Medium 2 contained 2500 mg / L KNO ₃ , 150 mg / L CaCl ₂ .2H ₂ O, 250 mg / l MgSO ₄ .7H ₂ O, 134 mg / l (NH ₄ ) ₂ SO ₄ , 150 mg / l NaH ₂ PO ₄ .H ₂ O, 0.75 mg / l KI, 3.0 mg / l H ₃ BO ₃ , 10 mg / l MnSO ₄ .H ₂ O, 2.0 mg / l ZnSO ₄ .7H ₂ O, 0.25 mg / l of Na ₂ MoO ₄ .2H ₂ O, 0.025 mg / l CuSO ₄ .5H ₂ O, 0.025 mg / l CoCl ₂ .6H ₂ O, 43 mg / l Ferric-EDTA, 2% sucrose, , inositol of 100 mg / l, nicotinic acid of 1.0 mg / l, pyridoxine (HCl) of 1.0 mg / l, thiamine (HCl) of 10 mg / / l of kinetin, 0.1 to 1.0 mg / l of 2,4-D,

Medium 3 contained 0.0025 mg / l of CoCl ₂ .6H ₂ O, 0.0025 mg / l of CuSO ₂ .5H ₂ O, 36.7 mg / l of FeNaEDTA, 10.62 mg / l of H ₃ BO ₃ , 1.58 mg / , 1.68 mg / l of MnSO ₄ .H ₂ O, 0.0025 mg / l Na ₂ MoO ₄ .2H ₂ O, 1.06 mg / l ZnSO ₄ .7H ₂ O, 226.50 mg / l CaCl ₂ , 600 mg / Of KNO ₃ , 244.33 mg / l of MgSO ₄ , 250 mg / l of NaH ₂ PO ₄ , 400 mg / l of (NH ₄ ) ₂ SO ₄ , 100 mg / l of myo-Inositol, , 1.0 mg / l of nicotinic acid, 1.0 mg / l of pyridoxine hydrochloride and 10.0 mg / l of thiamine hydrochloride,

Medium 4 contained 0.25 mg / l of CuSO ₄ .5H ₂ O, 36.7 mg / l of FeNaEDTA, 6.2 mg / l of H ₃ BO ₃ , 22.3 mg / l of MnSO ₄ .H ₂ O, 0.25 mg / Na ₂ MoO ₄ .2H ₂ O, 8.6 mg / l ZnSO ₄ .7H ₂ O, 72.50 mg / l CaCl ₂ , 471.26 mg / l Ca (NO ₃ ) ₂ .4H ₂ O, 170 mg / l KH ₂ PO ₄ , 990 mg / l K ₂ SO ₄ , 180.54 mg / l MgSO ₄ , 400 mg / l NH ₄ NO ₃ , 2.00 mg / l glycine, 100 mg / l myo-Inositol, 0.5 (Pyridoxine HCl) and 1.0 mg / l of thiamine hydrochloride (Thiamine HCl).

Medium 5 contained 2830 mg / L KNO ₃ , 463 mg / L ammonium sulphate, 166.02 mg / L CaCl ₂ .2H ₂ O, 90.37 mg / l MgSO ₄ , 400 mg / l KH ₂ PO ₄ , 3.00 mg / l of MnSO ₄ .H ₂ O, 1.60 mg / l of boric acid, 0.80 mg / l of KI, 1.5 mg / l of ZnSO ₄ .7H ₂ O, 37.26 mg / l of ferrous sulphate 7H ₂ O, 1.0 mg / l EDTA disodium salt · 2H ₂ O, 0.5 mg / l pyridoxine hydrochloride, 0.5 mg / l nicotinic acid, 2.0 mg / l glycine, Lt; / RTI >

Medium 6 is 0.03mg / ℓ of CuSO ₄ and 5H ₂ O, 36.70mg / ℓ of the _{FeNaEDTA, 10mg / ℓ H 3 BO} 3, 18.94mg / ℓ of MnSO ₄ and H ₂ O, Na ₂ of 0.25mg / ℓ MoO ₄ .2H ₂ O, 10 mg / l ZnSO ₄ .7H ₂ O, 166 mg / l CaCl ₂ , 68 mg / l KH ₂ PO ₄ , 950 mg / l KNO ₃ , 90.27 mg / l MgSO ₄ , / l of NH ₄ NO ₃ , 0.03 mg / l of biotin, 0.5 mg / l of folic acid, 2.0 mg / l of glycine, 100 mg / l of myo-inositol, , Nicotinic acid of 5.0 mg / l, 0.5 mg / l of pyridoxine hydrochloride and 0.5 mg / l of thiamine hydrochloride,

Medium 7 contained 0.025 mg / l CoCl ₂ .6H ₂ O, 0.025 mg / l CuSO ₄ .5H ₂ O, 36.7 mg / l FeNaEDTA, 6.2 mg / l H ₃ BO ₃ , 0.83 mg / , 16.90 mg / l of MnSO ₄ .H ₂ O, 0.25 mg / l of Na ₂ MoO ₄ .2H ₂ O, 8.6 mg / l of ZnSO ₄ .7H ₂ O, 270.00 mg / l of KH ₂ PO ₄ , a / ℓ of KNO3, 175.79mg / ℓ of _{MgSO 4, CaCl 2, 400mg /} ℓ of 166mg / ℓ NH ₄ NO _3, thiamine hydrochloride of myoinositol (myo-inositol), 0.4mg / ℓ of 100mg / ℓ ( Thiamine HCl)

Medium 8 contained 0.025 mg / l CoCl ₂ .6H ₂ O, 0.025 mg / l CuSO ₄ .5H ₂ O, 36.7 mg / l FeNaEDTA, 6.2 mg / l H ₃ BO ₃ , 0.08 mg / , 0.76mg / ℓ of _{MnSO 4 · H 2 O, 0.25mg} / ℓ of _{Na 2 MoO 4 · 2H 2 O} , 8.6mg / ℓ of _{ZnSO 4 · 7H 2 O, 578.92mg} / ℓ of Ca (No _{3) 2} Anhydrous, 270.00 mg / l KH ₂ PO ₄ , 1800 mg / l KNO ₃ , 175.79 mg / l MgSO ₄ , 400 mg / l NH ₄ NO ₃ , 100 mg / l myo-Inositol, 0.4 mg / l thiamine hydrochloride (Thiamine HCl)

Medium 9 is 0.001mg / ℓ of _{CuSO 4 · 5H 2 O, 3.47mg} / ℓ of _{FeSO 4 · 7H 2 O, 1.5mg} / ℓ of H ₃ BO _3, of 0.75mg / ℓ KI, of 5.31mg / ℓ MnSO ₄ H ₂ O, 0.0001 mg / l MoO ₃ , 200 mg / l Na ₂ SO ₄ , 2.67 mg / l ZnSO ₄ .7H ₂ O, 208.47 mg / l Ca (No ₃ ) ₂ anhydrous, 65 mg / ℓ, and the KCl, KNO _3, the medium consisting of NaH ₂ PO ₄ of MgSO _4, 16.8mg / ℓ of 351.6mg / ℓ of 80mg / ℓ,

Medium 10 contained 0.025 mg / l CoCl ₂ .6H ₂ O, 0.025 mg / l CuSO ₄ .5H ₂ O, 73.4 mg / l FeNaEDTA, 6.2 mg / l H ₃ BO ₃ , 0.3 mg / , 16.90 mg / l of MnSO ₄ .H ₂ O, 0.25 mg / l of Na ₂ MoO ₄ .2H ₂ O, 8.6 mg / l of ZnSO ₄ .7H ₂ O, 332.02 mg of CaCl ₂ , 480 mg / Of KNO ₃ , 180.54 mg / l of MgSO ₄ , 330.60 mg / l of NaH ₂ PO ₄ , and 400 mg / l of NH ₄ NO ₃ .

As shown in Fig. 1, from the results of Example 2, Medium 7, which is relatively small in micro-tuber formation, was selected as the optimal medium for the production of egg yolk. In the following Examples, Medium 7 The liquid medium or solid medium was used for the experiment.

Example  3. Depending on the type and concentration of plant growth regulators Contrary Everything Small tuber   Confirm formation

Auxin - based plant hormone was added additionally to investigate the effect on the root growth. Growth indices of barnyard tubers were determined by adding indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) to medium 7,

As a result, as shown in Figs. 2 and 3, it was confirmed that the growth index of the small tuber was increased by adding IBA and IAA. In particular, when indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) was added in an amount of 5.0 to 10.0 mg / l, .

In addition, the ability to differentiate tuberous tubers using plant growth regulators was compared according to sucrose concentration. As a result, it was confirmed that the 3% sucrose was basically added to provide carbohydrates. In the case of in vitro culture, 6% sucrose was treated to increase the small tuber formation rate and yield (FIG. 4).

In addition to auxin hormone, it regulates growth and promotes cell division. It acts as a dormant detergent, inhibits aging of plant tissues, promotes leaf and eyelid growth, and prevents aging of leaves and fruit. It can be promoted through combination with tokinin, and it has been confirmed that a combination of auxin plant growth regulators can induce multiple roots in a tissue having rootstock or differentiation ability (FIG. 5).

From Example 3, the conditions under which the small tuber (barnacles) under adverse conditions were produced were 6% sucrose, 1.0 mg / l benzyladenine (BA) and 1.0-5.0 mg / l IAA, and as a result, it was confirmed that 15 or more anti-alga can be obtained in one treatment (Fig. 6).

Example  4. Alban Ha  Purification and Grave  production

Based on the production of Albanha through optimal culture conditions in the cabin, the massive plants, that is, Seedling state, were named as seedling semi-summer. Based on the treatment of medium 7 and plant growth regulators (auxin and cytokinin: IAA and BA), which were established under the optimum culture conditions, the root enlargement effect and productivity, and the adaptation condition of the albaine to the soil can be confirmed by applying to the soil .

One of the issues to be considered in the latter part of establishing the standard growth method of nutrient-based standard for the production of standard seedlings is the adaptation of the actual meals to the outdoors environment and soil adaptation. Nutrient breeding through large-scale organisms can induce adaptation to the environment after the pretreatment of purification by the following method. This process is one of the development of the extra-purification technology of in-flight nutrients, and it can be said that it is seedling production through adaptation and acclimatization.

The mixed soil used in Example 4 was cultivated in a mixed soil of three kinds in order to compare the adaptation and growth of the cultivated seedlings cultivated in the in vitro culture medium. The mixed soil may be selected from the group consisting of Coir (Sun Cocopeat Co., Vietnam), Peat moss (Sun Gro Co., Canada), Vermiculite (Green fire chemicals Co., Korea), Perlite (Kyungdong ONE Co. , Korea) and zeolite (Zeolite, JinNong Co., Korea).

The soil C was mixed with coir, peat moss, vermiculite and zeolite in the same ratio as that of the commercially available soil. The soil A and B were used for the comparison of the growth depending on the moisture content and pore difference in the soil, The coir was mixed with 20% and 20% pearlite (Table 1). The pH of the supernatant was adjusted to 5.8, and a small amount of water was added during the mixing for 24 hours so as to be a sufficient function.

The composition ratio of the mixed soil
Growing media (% by weight) Topo A Base B Top soil C Coir 88 54 68 Peat moss 6 12 15 Vermiculite One 2 3 Perlite 3 27 7 Zeolite 3 5 7 Total 100 100 100

On the other hand, statistical analysis was performed to confirm the difference of soil growth by soil condition with and without solids during incubation. One-way analysis of variance (ANOVA) and Duncan's multiple test (α = 0.05) were performed using SPSS (SPSS 2.0.0, SPSS Inc., USA)

1) mixing Soot  Environmental adaptation by composition

After in vitro culture, weighed growth was carried out in various mixed soil compositions to compare soil mobilization. The leaves and stems of the harvested leaves were removed, and the seeds were pretreated by soaking in water for 3 days. Before planting in the soil, the pure tubers were weighed and the tubers were removed from the tubers for the purpose of constant regrowth conditions between the samples. The weight and size of the tubers were removed and replicated in four replicates of five each. The dry edges were excluded at the time of planting. Standard plate seedling planting is complete, each mixed bed soil (50 hole tray, Seoulbio, Korea ) , the temperature 22 ℃, brightness 67.5μmolm ^-2 S ^{- 1} and 16/8 photoperiod contrast cycle set saengyuksang (JSPG-1500C, JSR, Korea ), And the tubers were grown for 8 weeks (Fig. 7).

As a result, it was confirmed that the tubers produced by the in - cabinet culture needed soil purification and environmental adaptation for use as seedlings in farmhouses. The adaptation to the soil environment varies depending on the conditions applied in the incubation. It was confirmed that it is important to establish the culture conditions in the cabinet for the excellent soil mobilization of the barnyard tubers.

The environmental adaptation according to the soil purification rate and in - flight culture conditions was confirmed first. First, the tubers used in the experiment were grown in a growth chamber set at 25 ° C. After that, the surface of the tubers was observed at the 4th and 8th weeks to confirm the initial sticking of the tubers (Fig. 8) , G / tuber and net weight (%) were confirmed (Fig. 9).

Above ground showed the highest occurrence rate of 92% in soil B, 72% in soil A and 69% in soil C, respectively.

After surviving for 8 weeks in each soil, survival rate was found to be more than 96% in Soil B (1.3 times), followed by Soil B, Soil A, and Soil C (Table 2).

2) mixing Soot  Comparison of growth of underground and underground depending on conditions

After the tubers were re - planted in the soil, the appearance rate of the above - ground part was confirmed at 4th week. After 8 weeks of growth, growth was examined. Number of leaves, number of dead leaves, number of tuber, tuber size (tuber size, number of leaves) size). Fresh weights were measured before and after growing the tubers in the soil to determine the quantitative difference, and the dry weights were measured after all growth investigations. When measuring live weight and building weight, leaves, stems, and roots except tubers were removed and measured.

In order to investigate the difference in the differentiation and differentiation of the tubers according to the conditions of the mixed soil, three different soil cultivars were cultivated for 8 weeks And then examined for growth. Firstly, the production and the yield of the leaves are important for the utilization and the enlargement of the tubers. The number of leaves was 1.7 in soil B and 1.1 and 1.0 in soil A and soil C, respectively, indicating significant differences in soil B (Table 2).

The results of the growth test according to the types of medium and mixed soil Growth media plant height
(cm / plant) Fresh leaves
(ea / plant) Dead leaves
(ea / plant) Bulbil
(ea / plant) Survival rate (%) A 6.5 ± 0.16 a 1.1 ± 0.08ab 0.0 ± 0.02b 1.0 ± 0.08a 93.1 B 5.9 ± 0.16 a 1.7 ± 0.07a 0.2 ± 0.04b 1.2 ± 0.08a 96.2 C 5.9 ± 0.12 a 1.0 ± 0.09ab 0.1 ± 0.04b 0.8 ± 0.08a 84.6

The number of dry leaves was highest in soil B, and it was judged that the roughness root of the undergrowth was damaged by drying in the soil where the pearlite increased drainage (Table 2).

As a result, it showed the highest value showing the significant difference when growing in the midway and in the soil B, and this was the result of the accumulation of anabolic products (Table 3).

The results of the growth test according to the types of medium and mixed soil Growth media Tuber
(ea / plant) Tuber size
(cm ² / tuber) Fresh tuber yield
(g / pot) Dry tuber yield
(g / pot)
A 0.9 ± 0.02a 1.4 ± 0.08a 0.6 ± 0.04a 0.2 ± 0.00 c B 1.0 ± 0.02a 1.4 ± 0.08a 0.4 ± 0.02a 0.2 ± 0.00a C 0.9 ± 0.04a 1.5 ± 0.12a 0.7 ± 0.05a 0.2 ± 0.00bc

On the other hand, in the case of fresh weight, there was no significant change in the soil A from 0.6 ± 0.01 g × pot ^-1 before planting to 0.6 g in the soil, and slightly decreased or increased to 0.4 g and 0.7 g in the soil B and C, respectively. Therefore, it was judged from the present Example 4 that the cultivation conditions influenced the transformation rate when the plant produced through in-vitro culture was purified to the soil. Therefore, it was concluded that appropriate selection of soil was necessary for the rapid mobilization after transplanting into soil.

On the other hand, the survival rate of soil A and C was lower than that of soil B because of the characteristics of growth in soil with good water dripping ability. Respectively. In contrast, it was judged that the tuber was more vulnerable to humidification than the other root. In this Example 4, soil growth and environmental adaptability were confirmed according to the growth environment of the cabin, and the growths in various mixed soils were compared.

Claims (10)

0.0125 ~ 0.0375mg / ℓ of _{CoCl 2 · 6H 2 O, 0.0125} ~ 0.0375mg / ℓ of _{CuSO 4 · 5H 2 O, 18.35} ~ of FeNaEDTA, 3.1 ~ 12.4mg / ℓ of _{73.4mg / ℓ H 3 BO 3,} 0.415 1 to 16.6 mg / l KI, 8.45 to 33.8 mg / l MnSO ₄ .H ₂ O, 0.125 to 0.5 mg / l Na ₂ MoO ₄ .2H ₂ O, 4.3 to 17.2 mg / l ZnSO ₄ .7H ₂ O, 135 ~ 540mg / ℓ of KH ₂ PO _4, 900 ~ of 3600mg / ℓ of KNO _3, 87.895 ~ 351.58mg / ℓ of MgSO _4, 83 ~ 332mg / ℓ of _{CaCl 2, 200 ~ 800mg / ℓ} NH 4 NO ₃ , 50 to 200 mg / l of myo-Inositol and 0.2 to 0.8 mg / l of thiamine hydrochloride as active ingredients. The culture medium for in vitro culture for the production of egg yolk according to claim 1, further comprising an auxin-based plant growth regulator in addition to the active ingredient. 3. The method according to claim 2, wherein the auxin plant growth regulator is indole-3-acetic acid (IBA), indole-3-butyric acid (IBA), or IPA (beta-indole propionic acid) In-vitro culture medium. 4. The plant growth regulator according to claim 3, wherein the auxin plant growth regulator is indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) in an amount of 1.0 to 5.0 mg / In vitro culture medium for the production of egg yolk. The culture medium for in vitro culture for the production of egg yolk according to claim 1, further comprising an auxin-based plant growth regulator, cytokinin and sucrose, in addition to the active ingredient. 6. The method according to claim 5, wherein the auxin-based plant growth regulator is IAA and the cytokinin is BA and further comprises 3-6% (w / v) sucrose. A culture medium composition for culture. delete (1) A method for producing a microorganism, comprising the steps of: (a) directing an inferior turbinate to a culture medium containing the culture medium according to any one of claims 1 to 6 to induce multi-tuber formation; And
(2) a step of environmentally adapting the produced egg halves in a mixed soil containing a coir, peat moss, vermiculite, perlite and zeolite; A mass production method that is characterized by gravestones. 9. The method of claim 8, wherein the mixed soil comprises 48 to 60 wt% Coir, 10 to 14 wt% peat moss, 1.5 to 2.5 wt% vermiculite, 24 to 30 wt% By weight of perlite and 4 to 6% by weight of zeolite. delete

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