KR102154846B1 - Method for culturing plant tissue of Paeonia lactiflora Pall. - Google Patents

Abstract

The present invention relates to a plant culture method of peony. By establishing an optimal medium composition to form a plant of peony from a winter bud part of the peony and inducing shoot formation, multi-stem induction, and rooting in a medium having the optimum medium composition, peony with excellent survival rate and growth was proliferated. Therefore, the method according to the present invention can be usefully used for mass production of peony.

Description

Method for culturing plant tissue of Paeonia lactiflora Pall.}

The present invention relates to a method for culturing a plant of peony.

Peony (芍藥) is one of the very important herbal medicines as the root of the plant of the same genus. Leaves are alternate, petioles are long, exudate twice, small leaves are oval, and there are no saw teeth. The flowers are also called hampock flowers, one at the end of the stem, and white flowers bloom from May to June. The roots are dried with herbal medicine, and the dough grows in the shade of a mountain tree. It is distributed all over Korea, and in oriental medicine, the subterranean stems are collected in March and August, dried in the sun, and dried in the sun, alveolar, blood, blood, ikbi, and jitong. It is used for 止痛). In oriental medicine, it has been used as astringent relief (斂緩和), jingyeong (鎭痙), pain reliever (痛), and has been used as Gyejigahwagyun yaktang. Fruits are Goldolaceae, 1 to 3, with a horn-shaped red unmatured seeds and black mature seeds exposed. Peony used for medicinal purposes is a medicinal crop belonging to the genus Paeonia of Ranuculaceae, and is classified into peony, a herbaceous plant, and peony, a wooden tree. Peonies grown in Korea are classified into Paeonia lactiflora Pall, Paeonia obovata Max., and Paeonia japonica Miyabe et Takeda in terms of plant classification.

Despite the above-described usefulness, peony has a low seed germination rate, and as a propagation method, mainly by dispensing (giving away). However, dispensing can only be propagated once in spring and dispensing is possible for 2-3 weeks per plant, so mass production is difficult, and mass production technology through tissue culture is required.

On the other hand, the technology of culturing tissues such as eyes, leaves, stems, and roots of plants in an artificial in-flight environment to produce intact plants is called “tissue culture”. In a culture room where temperature and humidity are controlled, glass bottles or test tubes are used. It is a technology that puts nutrients that allow plants to grow in a culture vessel and grows plants in a sterile state without contamination. This tissue culture technology has the advantage of being able to produce seedlings all year round regardless of the season, so that when appropriate culture conditions are established, in-flight plants can be mass-produced at once.

Plants are composed of several types of organs, namely roots, stems, leaves, and firearms, and all organs have differentiation capabilities, but the differentiation capabilities differ depending on the type of organ, so the selection of an appropriate culture organ is very important. It is cultivated using the government (shoot tip), axillary (lateral eye, Axillary bud), and leaf segment (leaf segment). The unique differentiation ability of a plant is called totipotency, and it refers to the ability to regenerate a complete plant from a single cell or part of a plant tissue. Although all cells have proliferative ability, the results expressed differ significantly depending on the degree of differentiation of the cell tissue, the collection site, the composition of the medium, and the culture environment, so it is necessary to optimize the culture conditions according to the type of plant or variety. (Republic of Korea Patent Publication No. 10-2017-0122946).

In particular, various nutrients are required to grow and proliferate in a special environment where the tissues or organs of plants are in the cabin (in the state of a culture vessel such as a culture test tube or a glass bottle). However, the demand for nutrients depends on the type of plant or Since it varies according to the type, it is necessary to develop a medium suitable for the characteristics of the plant or tissue to be cultured. It was Murashige and Skoog (1962) that produced and developed a medium for plant tissue culture in earnest. This medium, called MS (Murashige & Skoog) medium, is a medium that balances inorganic salts, vitamins and energy sources. to be. In addition to MS medium, DKW medium, Gamborg B5 medium, White medium, WPM medium, etc. are widely used as basic medium for plant tissue culture. Based on this basic medium, the optimum medium composition must be selected and cultured according to the type of plant and the purpose of cultivation. In this case, in addition to the basic medium, specific inorganic salts, carbon sources, other organic substances, vitamins and growth regulators, etc., are added or concentration and partial It is necessary to change the content of the ingredients.

Accordingly, as a result of researching a method for mass propagation of peony, the inventors of the present invention established a method for efficiently proliferating peony through the rooting induction process after inducing shoot formation and multi-angularity by plaiting the Donga region of the peony where the growth point exists. By doing this, the present invention was completed.

An object of the present invention is to provide a method for culturing a plant of peony.

To achieve the above object,

The present invention comprises the steps of 1) inducing shoots by placing the dong-a portion of peony on a culture medium to which a growth regulator is added; 2) inducing multi-view from the induced shoots of step 1) using a culture medium to which a multi-view inducing agent is added; And 3) inducing rooting using a culture medium to which a rooting inducing agent is added from the induced multiplicity of step 2).

The present invention relates to a method for cultivating a plant of peony, and by establishing an optimal medium composition for forming a plant of a peony from the East Asian site of the peony, and inducing shoot formation, multi-cerebral induction, and rooting in a medium having the optimum medium composition, survival rate And a peony with excellent growth was proliferated. Therefore, the method according to the present invention can be usefully used for mass production of peony.

1 is a graph confirming the incidence of shoots from a region in which a growth point of a peony placed on MS medium or WPM medium exists.
Figure 2 is a graph confirming the length and number of shoots generated from the Dong-A site where the growth point of peony placed on MS medium or WPM medium is present.
FIG. 3 is a photograph of shoots induced from a region in which a growth point of peony placed on MS medium (A) or WPM medium (B) exists.
FIG. 4 is a graph confirming the incidence of shoots from the Dong-A site where the growth point of the dentate peony exists in MS medium to which zeatin and IBA, TDZ and IBA are added as growth regulators at various concentrations.
FIG. 5 is a graph confirming the length and number of shoots generated from the Dong-A site in which the growth point of the dentate peony exists in MS medium to which zeatin and IBA, TDZ and IBA are added as a growth regulator at various concentrations.
6 is a MS medium (control), MS medium (A) containing 2.0 mg/L of zeatin and 1.0 mg/L of IBA, and MS containing 2.5 mg/L of zeatin and 1.5 mg/L of IBA. Medium (B), MS medium (C) containing 3.0 mg/L zeatin and 2.0 mg/L IBA, MS medium (D) containing 0.15 mg/L TDZ and 1.0 mg/L IBA, The growth points of peony placed on MS medium (E) containing 0.20 mg/l of TDZ and 1.5 mg/l of IBA, and MS medium (F) containing 0.25 mg/l of TDZ and 2.0 mg/l of IBA This is a picture of a shoot derived from an existing Dong-A region.
FIG. 7 is a graph confirming the incidence of multi-view of peony from shoots placed on MS medium containing various concentrations of zeatin and IBA as a multi-viewer inducing agent, and mixed media of WPM and MS in which zeatin and TDZ are added in various concentrations.
FIG. 8 is a multi-diameter inducer confirming the multi-diameter length and number generated from shoots of peony placed on an MS medium containing zeatin and IBA at various concentrations, and a mixed medium of WPM and MS containing zeatin and TDZ at various concentrations It is a graph.
9 is a MS medium (control), MS medium (A) containing 2.0 mg/L of zeatin and 1.0 mg/L of IBA, and MS containing 2.5 mg/L of zeatin and 1.5 mg/L of IBA. Medium (B), MS medium (C) containing 3.0 mg/L zeatin and 2.0 mg/L IBA, WPM and MS containing 1.0 mg/L zeatin and 0.10 mg/L TDZ This is a photograph of the induction of polyhedra from the shoots of peony placed on the medium (D), a mixed medium (E) of WPM and MS containing 1.3 mg/ℓ of zeatin and 0.13 mg/ℓ of TDZ.
10 is a graph confirming the rooting rate from multiple diameters of peony placed on WPM medium to which IBA was added as a rooting inducing agent at concentrations of 0.5, 1.0, 1.5, and 2.0 mg/ℓ.
11 is a graph confirming the root length and number of roots generated from the multi-diameter of peony placed on WPM medium to which IBA was added as a rooting inducing agent at concentrations of 0.5, 1.0, 1.5, and 2.0 mg/ℓ.
FIG. 12 is a WPM medium (control), WPM medium containing 0.5 mg/L of IBA (A), WPM medium containing 1.0 mg/L of IBA (B), WPM medium containing 1.5 mg/L of IBA (C), a photograph of roots derived from the multi-diameter of peony placed on WPM medium (D) containing 2.0 mg/ℓ of IBA.
13 is a WPM medium (control), WPM medium containing 0.5 mg/L of IBA (A), WPM medium containing 1.0 mg/L of IBA (B), WPM medium containing 1.5 mg/L of IBA (C), a photograph of roots derived from the multi-diameter of peony placed on WPM medium (D) containing 2.0 mg/ℓ of IBA.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of 1) inducing shoots by placing the dong-a portion of peony on a culture medium to which a growth regulator is added; 2) inducing a multiplicity from the induced shoots of step 1) using a culture medium to which a multiplicity inducing agent is added; And 3) inducing rooting using a culture medium to which a rooting inducing agent is added from the induced multiplicity of step 2).

The method for culturing a plant of a peony according to the present invention provides a step of inducing shoots by placing the Donga region of the peony on a culture medium.

The Donga region may include a growth point.

As used herein, the term "culture medium" is a medium used in the step of inducing shoots, multi-plants or rooting from the Dong-A region of peony, and induces shoots from tissues of plants for tissue culture, or multi-plants from the resulting shoots. It contains a common basal medium that can be used to induce rooting or to induce rooting from the resulting multi-cerium. The culture medium may be artificially prepared and used, or a commercially available one may be used.

When the culture medium is prepared and used, the culture medium may contain any one or more selected from the group consisting of carbon sources, mass elements, trace elements, vitamins and amino acids.

An example of the composition of the culture medium is as follows.

The carbon source may be any one or more selected from the group consisting of sucrose, glucose, maltose, lactose, and fructose, and specifically sucrose. The carbon source may be included in a concentration of 20 to 40 g/ℓ, specifically 23 to 37 g/ℓ, and more specifically 26 to 33 g/ℓ.

The mass elements are MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, K ₂ SO ₄ , KCl, KI, KNO ₃ and Any one or more selected from the group consisting of NaH ₂ PO ₄ ·H ₂ O, specifically MgSO ₄ ·7H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, KCl, KI And any one or more selected from the group consisting of KNO ₃ , more specifically MgSO ₄ ·7H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, KCl, KI and KNO ₃ Can include.

The MgSO ₄ ·7H ₂ O may be included in a concentration of 25 to 45 mg/ℓ, specifically 30 to 40 mg/ℓ, and more specifically 33 to 37 mg/ℓ. The NH ₄ NO ₃ may be included in a concentration of 800 to 1,200 mg/ℓ, specifically 850 to 1,150 mg/ℓ, and more specifically 900 to 1,110 mg/ℓ, and the KH ₂ PO ₄ is 270 to 330 mg/ℓ , Specifically, it may be included in a concentration of 280 to 320 mg/ℓ, more specifically 290 to 310 mg/ℓ. In addition, the Ca(NO) ₂ ·4H ₂ O may be included in a concentration of 330 to 360 mg/l, specifically 340 to 355 mg/l, more specifically 345 to 350 mg/l, and the KCl is 25 to It may be included in a concentration of 40 mg/ℓ, specifically 30 to 45 mg/ℓ, and more specifically 35 to 40 mg/ℓ. In addition, the KI may be included in a concentration of 55 to 75 mg/l, specifically 60 to 70 mg/l, more specifically 62 to 68 mg/l, and the KNO ₃ is 800 to 1,200 mg/l, specifically It may be included in a concentration of 850 to 1,150 mg/ℓ, more specifically 900 to 1,110 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 2H ₂ O And any one or more selected from the group consisting of CoCl ₂ ·6H ₂ O, specifically 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 ·2H ₂ O and CoCl ₂ ·6H ₂ O.

The FeSO ₄ ·7H ₂ O may be contained in a concentration of 24 to 30 mg/l, specifically 25 to 29 mg/l, more specifically 26 to 28 mg/l, and the MnSO ₄ ·H ₂ O is 5 to It may be contained in a concentration of 7 mg/L, specifically 5.5 to 6.5 mg/L, and more specifically 5.8 to 6.2 mg/L. The ZnSO ₄ ·7H ₂ O may be included in a concentration of 2 to 6 mg/ℓ, specifically 2.5 to 5.5 mg/ℓ, and more specifically 3 to 5 mg/ℓ. The CuSO ₄ ·5H ₂ O may be included in a concentration of 0.01 to 0.04 mg/ℓ, specifically 0.015 to 0.035 mg/ℓ, more specifically 0.020 to 0.030 mg/ℓ, and the H ₃ BO ₃ is 1 to 3 mg /ℓ, specifically 1.2 to 2.8 mg/ℓ, more specifically 1.5 to 2.5 mg/ℓ, and the Na ₂ -EDTA is 25 to 50 mg/ℓ, specifically 30 to 45 mg/ℓ, More specifically, it may be included in a concentration of 35 to 40 mg/ℓ. In addition, the Na ₂ MoO ₄ ·2H ₂ O may be included in a concentration of 0.01 to 0.04 mg/l, specifically 0.015 to 0.035 mg/l, more specifically 0.020 to 0.030 mg/l, and the CoCl ₂ ·6H ₂ O may be contained in a concentration of 0.01 to 0.04 mg/ℓ, specifically 0.015 to 0.035 mg/ℓ, and more specifically 0.020 to 0.030 mg/ℓ.

The vitamins and amino acids are any one or more selected from the group consisting of glycine, inositol, nicotinic acid, pyridoxine and thiamine, specifically any one or more selected from the group consisting of inositol, nicotinic acid, pyridoxine and thiamine, and more specifically inositol, nicotinic acid. , Pyridoxine and thiamine. The inositol may be contained in a concentration of 0.1 to 3 mg/ℓ, specifically 0.5 to 2 mg/ℓ, and more specifically 0.8 to 1.5 mg/ℓ. The nicotinic acid may be contained in a concentration of 0.03 to 0.2 mg/l, specifically 0.05 to 0.15 mg/l, and more specifically 0.08 to 0.12 mg/l. The pyridoxine may be contained in a concentration of 0.03 to 0.2 mg/l, specifically 0.05 to 0.15 mg/l, more specifically 0.08 to 0.12 mg/l, and the thiamine is 0.1 to 3 mg/l, specifically 0.5 to 2 It may be included in a concentration of mg/ℓ, more specifically 0.8 to 1.5 mg/ℓ.

Another example of the composition of the culture medium is as follows.

The carbon source may be any one or more selected from the group consisting of sucrose, glucose, maltose, lactose, and fructose, and specifically sucrose. The carbon source may be included in a concentration of 20 to 40 g/ℓ, specifically 23 to 37 g/ℓ, and more specifically 26 to 33 g/ℓ.

The mass elements are MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, K ₂ SO ₄ , KCl, KI, KNO ₃ and Any one or more selected from the group consisting of NaH ₂ PO ₄ H ₂ O, specifically MgSO ₄ 7H ₂ O, CaCl ₂ 2H ₂ O, NH ₄ NO ₃ KH ₂ PO ₄ Ca(NO) ₂ Any one or more selected from the group consisting of 4H ₂ O and K ₂ SO ₄ , more specifically MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ May contain 4H ₂ O and K ₂ SO ₄ .

The MgSO ₄ ·7H ₂ O may be included in a concentration of 350 to 390 mg/ℓ, specifically 360 to 380 mg/ℓ, and more specifically 365 to 375 mg/ℓ. The CaCl ₂ ·2H ₂ O may be included in a concentration of 90 to 102 mg/l, specifically 92 to 100 mg/l, more specifically 94 to 98 mg/l, and the NH ₄ NO ₃ is 370 to 430 mg /ℓ, specifically 380 to 420 mg/ℓ, and more specifically 390 to 410 mg/ℓ. In addition, the KH ₂ PO ₄ may be included in a concentration of 140 to 200 mg/ℓ, specifically 150 to 190 mg/ℓ, more specifically 160 to 180 mg/ℓ, and the Ca(NO) ₂ ·4H ₂ O May be included in a concentration of 530 to 580 mg/ℓ, specifically 540 to 570 mg/ℓ, and more specifically 550 to 560 mg/ℓ. In addition, the K ₂ SO ₄ may be included in a concentration of 960 to 1020 mg/ℓ, specifically 970 to 1010 mg/ℓ, and more specifically 980 to 1000 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 2H ₂ O And any one or more selected from the group consisting of CoCl ₂ ·6H ₂ O, specifically 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 any one or more selected from the group consisting of Na ₂ MoO ₄ ·2H ₂ O, more specifically 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 ·2H ₂ O.

The FeSO ₄ ·7H ₂ O may be contained in a concentration of 24 to 30 mg/l, specifically 25 to 29 mg/l, more specifically 26 to 28 mg/l, and the MnSO ₄ ·H ₂ O is 19 to It may be included in a concentration of 25 mg/ℓ, specifically 20 to 24 mg/ℓ, and more specifically 21 to 23 mg/ℓ. The ZnSO ₄ ·7H ₂ O may be included in a concentration of 6 to 10 mg/ℓ, specifically 7 to 9.5 mg/ℓ, and more specifically 8 to 9 mg/ℓ. The CuSO ₄ ·5H ₂ O may be contained in a concentration of 0.1 to 0.4 mg/l, specifically 0.15 to 0.35 mg/l, more specifically 0.2 to 0.3 mg/l, and the H ₃ BO ₃ is 5 to 7 mg /L, specifically 5.5 to 6.8 mg/L, more specifically 6 to 6.5 mg/L, and the Na ₂ -EDTA is 25 to 50 mg/L, specifically 30 to 45 mg/L, More specifically, it may be included in a concentration of 35 to 40 mg/ℓ. In addition, the Na ₂ MoO ₄ ·2H ₂ O may be contained in a concentration of 0.1 to 0.4 mg/ℓ, specifically 0.15 to 0.35 mg/ℓ, and more specifically 0.2 to 0.3 mg/ℓ.

The vitamins and amino acids may include any one or more selected from the group consisting of glycine, inositol, nicotinic acid, pyridoxine and thiamine, specifically glycine, inositol, nicotinic acid, pyridoxine and thiamine. The glycine may be contained in a concentration of 1 to 3 mg/l, specifically 1.2 to 2.8 mg/l, more specifically 1.5 to 2.5 mg/l, and inositol is 50 to 200 mg/l, specifically 60 to 150 It may be included in a concentration of mg/ℓ, more specifically 80 to 120 mg/ℓ. The nicotinic acid may be contained in a concentration of 0.1 to 1.0 mg/l, specifically 0.2 to 0.8 mg/l, and more specifically 0.4 to 0.6 mg/l. The pyridoxine may be contained in a concentration of 0.1 to 1.0 mg/l, specifically 0.2 to 0.8 mg/l, more specifically 0.4 to 0.6 mg/l, and the thiamine is 0.1 to 3 mg/l, specifically 0.5 to 2 It may be included in a concentration of mg/ℓ, more specifically 0.8 to 1.5 mg/ℓ.

The culture medium may be adjusted to pH 5.0 to 7.0, specifically pH 5.2 to 6.2, more specifically pH 5.5 to 6.0.

Specific examples of the commercially available culture medium may be MS (Murashige and Skoog basal), WPM (woody plant medium) medium.

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.

As used herein, the term "growth regulator" refers to a substance included in the culture medium to promote induction of shoots from the tissues of plants. The growth regulator may include a cytokinin-based or auxin-based compound, and more preferably, a mixture of a cytokinin-based compound and an auxin-based compound. Specifically, the growth regulator may be zeatin, IBA (indole-3-butyricacid), or a mixture thereof, and more preferably a mixture thereof, which is 1.5 to 3.0 mg/L of zeatin in the culture medium and IBA may be contained in a concentration of 0.5 to 2.0 mg / ℓ.

In addition, the method for culturing a plant of a peony according to the present invention provides a step of inducing a multiplicity from the induced shoots using a culture medium to which a multiplicity inducing agent is added.

As used herein, the term "multiplicity inducing agent" refers to a substance included in the culture medium to promote the induction of multiplicity from the induced shoots. The multimodality inducing agent may include a cytokinin-based compound and an auxin-based compound. Specifically, the multiplicity inducing agent may be zeatin, IBA (indole-3-butyricacid), or a mixture thereof, more preferably a mixture of zeatin and IBA (indole-3-butyricacid), which is a culture medium Zeatin may be contained in a concentration of 1.5 to 3.0 mg/ℓ and IBA 0.5 to 2.0 mg/ℓ.

In addition, the method of culturing a plant of a peony according to the present invention provides a step of inducing rooting using a culture medium to which a rooting inducing agent is added from the induced multi-gyeong.

As used herein, the term "rooting inducing agent" refers to a substance included in the culture medium to promote induction of rooting from the induced multilateral roots. The rooting inducing agent may include a cytokinin-based or auxin-based compound, more preferably an auxin-based compound. Specifically, the rooting inducing agent may be IBA (indole-3-butyricacid), which is 0.1 to 3.0 mg/l, specifically 0.25 to 2.5 mg/l, more specifically 0.5 to 2.0 mg/l in the culture medium Can be included as

In a specific embodiment of the present invention, the present inventors collected a region of Donga where a growth point exists from Donga of a peony, and then selected a medium and a growth regulator for inducing shoots using this. As a result, compared to the WPM medium, the length of shoots derived from the Donga region of the peony placed on the MS medium was longer, showing relatively excellent growth, and it was confirmed that the MS medium is suitable for tissue culture of the peony (Table 2 and 1 to 3).

In addition, as a result of inducing shoots from the East Asian region of peony by adding zeatin and IBA, TDZ and IBA as growth regulators to the selected MS medium, MS with zeatin and IBA added to the MS medium with TDZ and IBA It was confirmed that the growth rate of shoots derived from the dong-a site placed on the medium was high, the length of shoots was long, and the number of shoots was large (see Table 3 and FIGS. 4 to 6).

In addition, the obtained shoots were placed in MS medium with zeatin and IBA added as a multiplicity inducing agent, and a mixed medium of WPM and MS with zeatin and TDZ added to confirm the multiplicity induction effect. As a result, zeatin and IBA were added. It was confirmed that the incidence rate of multi-diameter is the highest in MS medium, and the multi-diameter length and number are also excellent (see Table 4 and FIGS. 7 to 9).

In addition, as a result of checking the rooting induction effect by placing the obtained multi-branch on MS medium containing IBA as a rooting inducing agent by concentration, it was confirmed that the rooting rate, number of roots, and number of roots were excellent in the medium to which 1.5 mg/ℓ of IBA was added. (See Table 5 and FIGS. 10 to 13).

Therefore, from the above, shoots are induced in a culture medium containing a growth regulator using the Dong-A region where the growth point of peony is present, and the induced shoots are placed on a culture medium to which a multiplicity inducing agent is added to induce multiplicity, and the induced It can be seen that the above method of inducing rooting by placing the rootstock in a culture medium to which a rooting inducing agent is added can be usefully used for culturing a plant of a peony.

In addition, the present invention provides a peony plant cultured by the above method.

The tissue of the peony can be obtained according to a method having the characteristics as described above. As an example, the peony tissue may be derived from a Dong-A region where a growth point exists, and the induction may be performed by placing on a culture medium as described above containing a growth regulator. Through this, shoots can be obtained from the Donga site where the growth point of the peony exists, and from the obtained shoots, as described above, using a culture medium containing a multiplicity inducing agent, a multiplicity is induced, and the rooting inducing agent from the obtained multiplicity A peony plant from which rooting is induced can be obtained using the added culture medium.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

<Example 1> Preparation of tissue for cultivation of peony

Donga of a peony ( Paeonia lactiflora Pall.) was collected from a peony plantation in the mountain 1-3, Miwon-ri, Miwon-myeon, Sangdang-gu, Cheongju-si, Chungcheongbuk-do.

Specifically, after collecting peony Donga, it was washed 2-3 times with running water, put in 70% ethanol and sterilized for 3 minutes, and then washed 3 times with distilled water. The peony with the growth point prepared as described above was put in a 70 mL 2.5% sodium hypochlorite solution in a clean bench, 2 to 3 drops of Tween 20 were added, sterilized for 30 minutes, and washed 5 times with sterile water. Ready.

<Example 2> Selection of medium for induction of shoots of peony

Using MS (Murashige and Skoog basal) medium and WPM (woody plant medium) medium, shoots were cultivated from Dong-A having a growing point prepared in Example 1.

Specifically, the MS or WPM medium having the composition shown in Table 1 below was adjusted to pH 5.8, and then 8% agar was added. The medium to which the agar was added was dispensed into a test tube by 8 ml, and then sterilized at 121° C. for 15 minutes using an autoclave to prepare a solid medium. Incubation chamber conditions were incubated at 23 ± 5 °C for 16 hours in a light of 2000 lux.

Composition (mg/ℓ) MS WPM Mass element MgSO ₄ 7H ₂ O 35 370 CaCl ₂ 2H ₂ O - 96 NH ₄ NO ₃ 1,000 400 KH ₂ PO ₄ 300 170 Ca(NO) ₂ 4H ₂ O 347 556 K ₂ SO ₄ - 990 KCl 37.3 - KI 65 - KNO ₃ 1,000 - NaH ₂ PO ₄ H ₂ O - - Trace elements FeSO ₄ 7H ₂ O 27.8 27.8 MnSO ₄ H ₂ O 6.06 22.3 ZnSO ₄ 7H ₂ O 4.0 8.6 CuSO ₄ 5H ₂ O 0.025 0.25 H ₃ BO ₃ 2 6.2 Na ₂ -EDTA 37.3 37.3 Na ₂ MoO ₄ 2H ₂ O 0.025 0.25 CoCl ₂ 6H ₂ O 0.025 - Etc Glycine - 2.0 Inositol 1.0 100 Nicotinic acid 0.1 0.5 Pyridoxine _· HCl (Pyridoxine _· HCl) 0.1 0.5 Thiamine _· HCl (Thiamine _· HCl) 1.0 1.0 Sucrose 30,000 30,000 Agar 8,000 8,000 pH 5.8 5.8

Donga having a growth point in the prepared solid medium was placed on the ground and observed for 4 weeks to investigate the incidence of shoots, the length of shoots, and the number of shoots, and are shown in Table 2 and FIGS. 1 to 3.

Badge Tooth
Number of population (pcs) New
The number of occurrences (pcs) Shoot incidence rate
(%) Shoot length
(mm) Number of shoots
(dog) MS 9 2 22.2 6.4±1.9 1.0±0.0 WPM 9 2 22.2 2.6±0.1 1.0±0.0

As a result, from 1 week after placement on the medium, the dong-a's seal leaves turned green, and shoots emerged from all of the dong-a placed on MS and WPM media from 3 weeks after placement. The shoot incidence rate in MS medium and WPM medium was the same as 22.2%, and the number of shoots was the same as 1.0±0.0, but the shoot length in MS medium was 6.4±1.9 mm, which was relatively superior to the shoot length of WPM medium, 2.6±0.1. .

Therefore, it was confirmed from the above that when the MS medium was used, shoots could be effectively generated from peony dong-a with a growing point.

<Example 3> Selection of growth regulators for induction of shoots of peony

In the process of culturing peony shoots using MS medium, the following experiment was performed to select an appropriate growth regulator.

Specifically, in order to select a growth regulator suitable for induction of shoots using sterilized and water-washed peony Donga as an experimental material, zeatin (6-[ 4-hydroxy-3-methyl-2-butenyl-aminopurine) and IBA (indole-3-butyric acid), a growth regulator of the auxin family, were respectively used in concentrations (2.0, 2.5, 3.0mg/L), (1.0, 1.5, 2.0mg/L) was added in combination, and the cytokinin TDZ (Tdiazurone) and the auxin-based IBA were added at each concentration (0.15, 0.2, 0.25mg/L), (1.0, 1.5, 2.0mg/L) The incidence rate of shoots and the length and number of shoots according to the treatment groups observed for 4 weeks by placing peony dong-a with a growing point by mixing with each other are shown in Table 3 and FIGS. 4 to 6.

Growth regulator (mg/L) Tooth
Population
(dog) New
The number of occurrences
(dog) Shoot incidence rate
(%) Shoot length
(mm) Number of shoots
(dog) MS
(0mg/L, control) 9 2 22.2 5.8±5.1 1.0±0.0 Zeatin + IBA
(2.0mg/L)(1.0mg/L) 9 5 55.6 15.4±6.8 1.0±0.0 Zeatin + IBA
(2.5mg/L)(1.5mg/L) 9 4 44.4 13.5±12.3 3.0±1.6 Zeatin + IBA
(3.0mg/L)(2.0mg/L) 9 4 44.4 5.3±2.9 2.8±2.4 TDZ+IBA
(0.15mg/L)(1.0mg/L) 9 2 22.2 11.3±7.4 1.5±0.7 TDZ+IBA
(0.2mg/L)(1.5mg/L) 9 3 33.3 6.8±0.4 1.0±0.0 TDZ+IBA
(0.25mg/L)(2.0mg/L) 9 One 11.1 10.9±0.0 1.0±0.0

As a result, the shoot incidence rate in MS medium without the addition of the growth regulator was 22.2%, the average shoot length was 5.8±5.1 mm, and the average shoot number was 1.0±0.0.

The highest shoot incidence rate was shown at 55.6% in the medium containing 2.0 mg/L of zeatin and 1.0 mg/L of IBA in MS medium, with the shoot length of 15.4±6.8 mm and the number of shoots of 1.0±0.0. Next, the medium with excellent results was a medium in which 2.5mg/L of zeatin and 1.5mg/L of IBA were mixed, and the shoot incidence rate was 44.4%, the shoot length was 13.5±12.3 mm, and the number of shoots was 3.0±1.6. Next, the shoot incidence rate was 44.4%, the shoot length was 5.3±2.9 mm, and the number of shoots was 2.8±2.4 with zeatin 3.0mg/L and IBA 2.0mg/L. It was found that the shoot incidence rate was better in the medium containing zeatin and IBA than the medium containing mixed TDZ and IBA.

Therefore, in the case of using a medium mixed with the cytokinin-based growth regulator zeatin 2.0mg/L and the auxin-based growth regulator IBA 1.0mg/L concentration, shoots can be effectively generated from peony dong-a with a growth point. It was confirmed that it can be.

<Example 4> Selection of multi-view inducing agents for multi-view induction from peony shoots

The medium for inducing multi-gyeong from the induced peony shoots was selected in the following manner.

Specifically, zeatin (6-[4-hydroxy-3-methyl-2-butenylamino-), a cytokinin system, in MS medium to which a growth regulator was not added for efficient multipathy induction in peony shoots under the conditions shown in Table 4 below. purine) and the growth control IBA (indole-3-butyric acid) of the auxin system were added at each concentration, and cytokinin zeatin and TDZ were added as a growth regulator in a mixture of ½ WPM medium and ½ MS medium by concentration. It was added mixed. The shoots induced through Dong-A culture were placed on each of the mediums and observed for 4 weeks, and the incidence of multi-diameter and the length and number of multi-diameters according to the medium are shown in Table 4 and FIGS. 7 to 9.

Growth regulator (mg/L) Tooth
Population
(dog) Multilateral occurrence
Population
(dog) Multilateral incidence rate
(%) Multi-diameter length
(mm) Polygyeongsu
(dog) MS(0mg/L, control) 9 One 11.1 3.4±0.0 1.0±0.0 Zeatin + IBA
(2.0mg/L)(1.0mg/L) 9 4 44.4 6.1±5.7 3.5±1.3 Zeatin + IBA
(2.5mg/L)(1.5mg/L) 9 4 44.4 14.7±13.2 2.0±0.8 Zeatin + IBA
(3.0mg/L)(2.0mg/L) 9 3 33.3 13.8±6.3 3.7±1.5 1/2MS+1/2WPM+zeatin+TDZ
(1.0mg/L)(0.1mg/L) 9 2 22.2 2.9±0.5 1.0±0.0 1/2MS+1/2WPM+zeatin+TDZ
(1.3mg/L)(0.13mg/L) 9 2 22.2 12.7±4.0 3.5±2.1

As a result, first, in the MS medium without the addition of a growth regulator as a control, the incidence rate of multi-diameter was 11.1%, the average multi-diameter length was 3.4±0.0 mm, and the average multi-diameter number was 1.0±0.0.

In MS medium, the number of polyhedritis occurred in the medium mixed with 2.0 mg/L of zeatin and 1.0 mg/L of IBA was an average of 3.5±1.3, resulting in the proliferation of the multilateral stem, and the multilateral incidence rate was 44.4%. Showed. Next, the number of polyhedra generated in the medium containing 3.0 mg/L of zeatin and 2.0 mg/L of IBA was an average of 3.7±1.5, the average length of the polyheal was 13.8±6.3 mm, and the incidence of polyheal was 33.3%. And multi-diameter lengths were the best, but the incidence rate of multi-diameters was low, showing the second excellent result. Next, in the medium containing 2.0 mg/L of zeatin and 1.5 mg/L of IBA, the average number of polyethers was 2.0±0.8, the average length was 14.7±13.2 mm, and the incidence rate was 44.4%, while ½ WPM. In the medium mixed with medium and ½ MS medium, in which cytokinin zeatin and TDZ as growth regulators were added at each concentration, the incidence of polymorphism was only 22.2%, indicating that it was not suitable for in-flight culture of peony.

Therefore, from the above, it was confirmed that the most suitable medium for multi-light induction of peony is to add 2.0 mg/L of the growth regulator zeatin and 1.0 mg/L of IBA to MS medium.

<Example 5> Selection of a rooting inducing agent for rooting induction from multiple roots of peony

The medium and the rooting inducing agent for inducing rooting from the multi-diameter base of the peony derived in Example 4 were selected in the following manner.

Specifically, the growth regulator IBA (indole-3-butyric acid) of the auxin family was treated as a single dose (0.5, 1.0, 1.5, 2.0 mg/L) as a growth regulator in the WPM medium to which the growth regulator was not added. The multi-diameter base of the peony was cut in each of the media and observed for 4 weeks after placing it in a test tube, and the rooting rate, root length and number of roots according to the media are shown in Table 5 and FIGS. 10 to 13.

Growth regulator (mg/L) Tooth
Population
(dog) Root
Occur
Population
(dog) Rooting rate
(%) Root length
(mm) Number of roots
(dog) WPM(0mg/L, control) 15 5 33.3 23.4±13.0 1.8±1.3 WPM+IBA(0.5mg/L) 15 6 40.0 9.5±7.9 3.5±2.4 WPM+IBA(1.0mg/L) 15 10 66.7 11.1±6.3 7.5±8.6 WPM+IBA(1.5mg/L) 15 12 80.0 11.7±5.8 9.2±5.1 WPM+IBA(2.0mg/L) 15 7 46.6 11.5±12.0 9.0±1.9

As a result, rooting began to be induced in the medium to which IBA was added to the WPM medium by concentration after 4 weeks on the medium, whereas rooting started from 6 weeks after the tooth in the WPM medium. The medium that showed the best results was a medium containing IBA 1.5mg/L added to WPM medium, showing 80% rooting rate, average root length of 11.7±5.8 mm, and average number of roots 9.2±5.1, making it the most suitable medium for inducing rooting of peony. appear. Next, the medium with IBA 1.5mg/L added to the WPM medium showed the second best results with a rooting rate of 66.7%, an average root length of 11.1±6.3 mm, and an average number of roots of 7.5±8.6. On the other hand, in the WPM medium without the addition of the growth regulator, the rooting rate was 33.3%, and the average number of roots was 1.8±1.3.

Claims (11)

1) Zeatin (Zeatin; 6-[4-hydroxy-3-methyl-2-butenyl-aminopurine) at a concentration of 1.5 to 3.0 mg/L and IBA (indole-) at a concentration of 0.5 to 2.0 mg/L. 3-butyric acid) inducing shoots by placing on a culture medium to which a shoot-inducing agent is added;
2) From the shoots induced in step 1), a multi-diameter inducing agent, which is a mixture of zeatin at a concentration of 1.5 to 3.0 mg/l and indole-3-butyric acid (IBA) at a concentration of 0.5 to 2.0 mg/l, is added. Inducing multi-gyeong using the MS culture medium; And
3) The step of inducing rooting using a WPM (Woody plant medium) culture medium added with an IBA (indole-3-butyric acid) at a concentration of 0.5 to 2.0 mg/L from the induced multi-diameter of step 2) A method of culturing a plant containing peony.
The method of claim 1, wherein the Dong-A region includes a growth point.


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