KR102200064B1 - Method of producing virus free M.9 and M.26 dwarf apple tree rootstock using apical meristem culture - Google Patents

Abstract

The present invention relates to a method for producing virus-free M.9 and M.26 dwarf apple rootstocks using growing point culture, which comprises the following steps: collecting growth points from axillary buds of M.9 or M.26, which are dwarf apple rootstock varieties, and placing the collected growth points on a first Murashige and Skoog basal (MS) medium including 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), and glycine to grow shoots; culturing the grown shoots in a second MS medium containing BAP, IBA, glycine, and glucose to differentiate them into seedlings; culturing the seedlings in a third MS medium containing IBA to induce rooting; and externally acclimatizing the rooting-derived seedlings. According to the present invention, since the appropriate medium and culture conditions for each culturing step are used, a better shoot formation rate, shoot growth, and rooting rate, and a lower browning rate than those of a case of performing cultivation in a basic medium, and thus the method can be usefully used for in-vitro mass proliferation of apple dwarf rootstocks.

Description

Method of producing virus free M.9 and M.26 dwarf apple tree rootstock using apical meristem culture using growth point culture

The present invention relates to a method for producing virus-free strains of apple dwarf stocks M.9 and M.26 using growth point culture.

The apple tree ( Malus pumila Miller ) is one of the major orchard crops and accounts for 23% of the total fruit production, and its cultivation area is increasing due to the recent strong apple prices and local government support when creating new orchards. In addition, the need for quality apple seedlings is also increasing, and the production of apple seedlings is recognized as a high value-added industry in the agricultural field. However, perennial fruit tree seedlings, including apples, have been propagated by breeding and seeding for 2-3 years, but the series is difficult due to the base phenomenon, and it is very difficult to secure a large-scale nursery that needs to be moved to industrialization and urbanization. It is small. In addition, most fruit tree seedling cultivation methods in Korea are cultivated in the open field, not in facilities, and thus are significantly affected by the environment. In addition, since apple seedlings are propagated using grafting, large trees and reception are required, and since it is difficult to cut apples, it is difficult to secure uniform stocks. Currently, the central fruit tree seedling management center produces and supplies disease-free stocks, but the production volume is so insufficient that it is impossible to secure the desired quantity, and the production rate and distribution rate of disease-free seedlings are very low.

On the other hand, the apple virus greatly affects the growth, production, and quality, and the Agricultural Cooperative Federation established the Central Fruit Tree Seedling Management Center with support for agricultural food in Korea, and is striving to supply disease-free seedlings. . Since the large-scale disease-free seedling breeding system has not been established, large-scale seedlings are imported from the Netherlands to breed and disease-free seedlings have been secured, but the production is insufficient, which does not greatly meet the request from the seedling industry.

When a plant is infected with a virus, the quality and quantity will drop by more than 30-50%, but since there is no direct treatment method, it is currently the most effective method to cultivate disease-free seedlings through apical division (growth point) culture and use it. Therefore, in apples, growth point cultivation has been conducted in order to obtain non-toxic seedlings. Most of the viruses present in the apple tree move through the catheter of the neck tissue, but the duct is not developed in the jugular area where the growth point is distributed, so the virus cannot penetrate. Therefore, in order to produce a healthy apple tree that is not diseased, the growth point or the stage of cultivation must be preceded. At this time, since the size of the cut growth point is as small as 0.1±0.3mm, the survival rate is very low. Therefore, the development of a medium to increase the survival rate of this growth point can be said to be a very important factor in the method of producing healthy seedlings. In addition to growing point culture, in-flight leaf section culture is also one of the important methods for removing viruses. Most of the viruses move along the duct part of the stem, so inducing callus from the leaf slice and re-differentiating the plant from it has the effect of blocking the infection of the virus moving along the duct. However, there is currently a need to develop a new technology for the effective mass growth of apple seedlings.

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 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. Therefore, it is very important to find a tissue culture medium composition suitable for M.9 and M.26 stocks for mass propagation of apple dwarf stocks and production of young plants.

As a prior literature on the composition of a medium for mass production of apple dwarf stocks, there is a document that discloses a method for mass production of dwarf stocks using ascorbic acid and citric acid as antioxidants, IBA and GA3 as plant growth regulators, and sucrose as a carbon source ( Journal of Plant Tissue Culture, Vol. 28, No. 1 33-36 (2001)), Korean Patent No. 10, which discloses a medium for mass production of apples with MS medium containing cytokinin as a growth regulator and sucrose as a carbon source. No. -1641301 exists, but the literature does not disclose a medium using a combination of IBA and BAP as a growth regulator and glycine as an antioxidant.

Accordingly, the present inventors cultured the growth point in a basic 4.4g/L MS medium, in which a growth regulator BAP 1mg/L, IBA 0.2 mg/L, an antioxidant glycine 4mg/L and a carbon source glucose 30g/L were further added. The present invention was completed by confirming that when an apple dwarf stockpile non-toxic seedling was produced by the method, the apple plant growth characteristics were excellent, and it was possible to mass-produce the virus-free apple dwarf stockpile virus-free stock.

It is an object of the present invention to provide a tissue culture method suitable for mass propagation of non-toxin seedlings of apple dwarf stocks M.9 and M.26 varieties using growth point culture.

In order to achieve the above object, the present invention is to collect a growth point from an Axillary bud of an apple dwarf stock variety, M.9 or M.26, and the collected growth point is BAP (6-benzylaminopurine), IBA (Indole-3). -butyric acid), glycine (Glycine) and glucose (Glucose) containing the first MS (Murashige and Skoog basal) the step of growing shoots by placing on the medium; Culturing the grown shoots in a second MS medium containing BAP, IBA, glycine and glucose to differentiate into young plants; Inducing rooting by culturing the young plant in a third MS medium containing IBA (Indole-3-Butyric Acid); And purifying the young plant from which the rooting has been induced. It provides a method for producing virus-free strains of M.9 or M.26, which are apple dwarf stocks including a.

The method for producing non-toxin seedlings of apple dwarf stock M.9 and M.26 varieties using the growth point culture of the present invention uses a medium and culture conditions suitable for each cultivation step, so that the survival rate, shoot formation rate, shoot shoots compared to the case of culturing in a basic medium Since the growth and rooting rate is excellent and the browning rate is low, the method for producing non-poisonous seedlings of the present invention can be usefully used for mass propagation of apple dwarf stocks.

1 is a photograph of growth point culture according to the present invention.
2 is a photograph of a shoot cultured according to the present invention.
3 is a photograph of the cultured young plant according to the present invention.
4 is a photograph of rooting culture according to the present invention.
5 is a photograph of the out-of-air purification according to the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, the present invention collects a growth point from an Axillary bud of an apple dwarf stock variety, M.9 or M.26, and the collected growth point is BAP (6-benzylaminopurine), IBA (Indole-3-butyric acid) , Glycine (Glycine) and glucose (Glucose) containing the first MS (Murashige and Skoog basal) step of growing shoots by placing on the medium; Culturing the grown shoots in a second MS medium containing BAP, IBA, glycine and glucose to differentiate into young plants; Inducing rooting by culturing the young plant in a third MS medium containing IBA (Indole-3-Butyric Acid); And purifying the young plant from which the rooting has been induced. It provides a method for producing virus-free strains of M.9 or M.26, which are apple dwarf stocks including a.

In the method for producing a disease-free apple dwarf stock of the present invention, the medium in each step may be used as a basic medium of MS (Murashige & Skoog) medium.

The MS (Murashige & Skoog) medium is a medium widely used as a basic medium when culturing plant tissues, and is characterized by a higher ratio of NH ₄ NO ₃ (20.6mM) and KNO ₃ (18.8mM) compared to other mediums. The MS medium contains more nitrate nitrogen as the mole ratio of nitrate nitrogen/ammonia nitrogen is about twice, and the total nitrogen content is 60 mM, which is significantly higher than that of other mediums. Based on the basic medium, the optimum medium composition can be selected by changing the concentration and the content of some components according to the type of plant and the purpose of cultivation. At this time, the composition of which the addition and content is changed includes specific inorganic salts, carbon sources, other organic substances, vitamins, and growth regulators, but is not limited thereto.

Various types of saccharides added to the MS medium act as energy sources of cultured tissues that do not have the ability to synthesize carbohydrates, and types of sugars used in tissue culture include sucrose, glucose, fructose, and Maltose and the like, but are not limited thereto. Among them, sucrose is dissolved in water and decomposed into monosaccharides glucose and fructose in the process of autoclaving, and invertase (reverse transcriptase) released from cell membranes of cultures is converted into monosaccharides, fructose. It is most widely used because it is hydrolyzed by an enzyme that hydrolyzes into glucose and glucose) or an extracellular enzyme, and changes into a form that is easy for the culture to absorb. However, the concentration of sucrose added to the basic medium may vary depending on the culture stage and the composition of the medium.

In the method for producing a disease-free apple dwarf stock of the present invention, specifically, in each step, the medium uses MS medium as a basic medium, and may further include a growth regulator, vitamins, etc., but is not limited thereto.

Vitamin B complexes such as Thiamine HCl (Thiamine HCI), Nicotinic acid (Nicotinic acid), Pyridoxine HCl (Pyridoxine HCl), riboflavin (Riboflabin), and Inositol (Inositol) are the vitamins necessary for tissue culture of plants. Can promote. In addition, ascorbic acid and citric acid inhibit the action of phenolic compounds generated in cultures, thereby reducing browning or blacking of cultured plants and increasing the production efficiency of healthy cultured seedlings. Browning or blacking that may occur during the process of plant tissue culture can occur when phenolic compounds generated in the culture are accumulated in the medium, the acidity of the medium decreases rapidly, and the nutrient absorption capacity of the medium transferred to the culture decreases. The above problem can be solved by frequently changing the culture medium to a new medium and culturing it again) or by adding an additive such as ascorbic acid, citric acid, or activated carbon to the medium.

Plant growth regulators are a generic term for plant hormones involved in plant growth, and even when plant tissue is cultured, only when a growth regulator of an appropriate type and concentration is added to the medium, the culture can grow and differentiate according to the purpose of culture. . The types of plant growth regulators include Auxins, Cytokinins, Gibberellin, Abscisic acid, but if plant hormones are involved in plant growth, plant formation of the present invention It may be included in the medium used in the method.

The auxin can be used primarily to induce root formation during tissue culture. Types of auxin include NAA (Naphthaleneacetic acid), IAA (3-Indole-acetic acid), IBA (Indole-3-butyric acid), and 2,4-D (2,4-dichlorophenoxyacetic acid). acid, 2,4-dichlorophenoxyacetic acid), and the like, but are not limited thereto.

Cytokinins are a derivative of adenine, one of the purine bases constituting nucleic acids, and mainly stimulate and promote cell division. When plant tissue is cultured, since the growth reaction is different for each type of plant according to the type and concentration of cytokinin, it can be used by adjusting the appropriate addition conditions in the medium. Cytokinins include kinetin, zeatin, BA (benzyladenine), BAP (6-benzylaminopurine, 6-benzylaminopurine, 6-BAP), TDZ (thidiazuron) and diphenylurea ( diphenyl urea) or the like may be used, but is not limited thereto.

The gibberellins may act on the kidney (stem elongation) of a plant, seed germination, flowering, fruiting, and fruit growth promotion. When a plant tissue culture is added with an appropriate concentration of gibberellin, seed germination and eye development can be promoted, and the stem of the cultured plant can be elongated to increase the height.

PPM (Plant Preservative Mixture, Plant Cell Technology) is an isothiazolone-based antibacterial agent that inhibits the enzymatic activity of various pathogens. It exhibits antibacterial activity by reacting specifically with the Krebs cycle and electron transport system in the respiratory matrix of microorganisms. In particular, since it is stable against heat, it can be autoclaved and can be used in the industrial culture process of various plants. By adding PPM to the medium, it is possible to reduce the incidence of contamination by microorganisms such as airborne bacteria or falling bacteria without showing any phytotoxicity to the plant being cultured.

In the disease-free production method of apple dwarf stocks of the present invention, the first MS medium is in MS medium at a concentration of 4 to 5 g/L, BAP 0.8 to 1.2 mg/L, IBA 0.1 to 0.5 mg/L, glycine 2 to 6 mg/L, glucose 25 to 35 g/L, and preferably in MS medium at a concentration of 4 to 5 g/L, BAP 0.8 to 1.2 mg/L, IBA 0.1 to 0.5 mg/L, glycine 2 To 6 mg/L, and 25 to 35 g/L of glucose.

The MS (Murashige & Skoog) medium is a medium widely used as a basic medium when culturing plant tissues, and is characterized by a higher ratio of NH ₄ NO ₃ (20.6mM) and KNO ₃ (18.8mM) compared to other mediums. The MS medium contains more nitrate nitrogen as the mole ratio of nitrate nitrogen/ammonia nitrogen is about twice, and the total nitrogen content is 60 mM, which is significantly higher than that of other media. Based on the basic medium, the optimum medium composition can be selected by changing the concentration and the content of some components according to the type of plant and the purpose of cultivation. At this time, the composition of which the addition and content is changed includes specific inorganic salts, carbon sources, other organic substances, vitamins, and growth regulators, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors used MS medium widely used as a basic medium when culturing plant tissues to establish a basic medium condition optimized for induction of plant shoots from the growth point of an apple dwarf stock, As a result of comparing the WPM medium developed for shoot cultivation by concentration, it was confirmed that the growth characteristics were better when MS medium was treated than WPM medium in both M.9 and M.26 varieties.

Plant growth regulators are a generic term for plant hormones involved in plant growth, and even when plant tissue is cultured, only when a growth regulator of an appropriate type and concentration is added to the medium, the culture can grow and differentiate according to the purpose of culture. . The types of plant growth regulators include Auxins, Cytokinins, Gibberellin, Abscisic acid, but if plant hormones are involved in plant growth, plant formation of the present invention It may be included in the medium used in the method.

The auxin can be used primarily to induce root formation during tissue culture. Types of auxin include Naphthalene acetic acid (NAA), 3-Indole-acetic acid (IAA), Indole-3-butyric acid (IBA), and 2,4-D (2,4- dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid), and the like, but are not limited thereto.

Cytokinins are a derivative of adenine, one of the purine bases constituting nucleic acids, and mainly stimulate and promote cell division. When plant tissue is cultured, since the growth reaction is different for each type of plant according to the type and concentration of cytokinin, it can be used by adjusting the appropriate addition conditions in the medium. Cytokinins include kinetin, zeatin, BA (benzyladenine), BAP (6-benzylaminopurine, 6-benzylaminopurine, 6-BAP), TDZ (thidiazuron) and diphenylurea ( diphenyl urea) or the like may be used, but is not limited thereto.

In a specific embodiment of the present invention, in order to confirm the plant growth regulator treatment conditions optimized for the induction of plant shoots from the growth point of the apple dwarf stock, each of the BAP 1mg/L, IBA 0.2mg/L, kinetin 0.5mg /L·IBA 0.2mg/L, GA3 0.5 mg/L·IBA 0.2mg/L, TDZ 0.5 mg/L·IBA 0.2mg/L and BAP 2mg/L with IBA 0.2mg/L, kinetin 0.5mg/L· Compared with the addition of 0.2 mg/L of IBA, 0.5 mg/L of GA3, 0.2 mg/L of IBA, and 0.2 mg/L of TDZ and 0.5 mg/L of IBA, the number of shoots in BAP 1 mg/L and IBA 0.2 mg/L treatment It was confirmed that the growth characteristics were the best with 2.3~2.6 pcs/week, shoot length 6.4~8.2cm, and survival rate 97.4~100%.

Browning or blackening is known to occur when phenolic compounds generated in the culture medium being cultivated in the culture vessel accumulate in the medium, sharply lowering the pH of the medium, and decreasing the absorption of nutrients in the medium transferred to the culture medium. . Plants cultured in-flight are artificially controlled and grown within a limited environment, and thus may receive various physiological stresses depending on the culture environment. It is known that phenolic compounds are generated from cultured plants by such stress factors. Therefore, it is possible to cultivate healthy plants only when the generation of these phenolic compounds is suppressed or the action is well controlled. In order to solve the problem of cultivation caused by phenolic compounds, subculture (changing the culture medium to a new medium and culturing it again) is frequently performed, or an additive such as activated charcoal or ascorbic acid is added to the medium. There is this. Ascorbic acid is vitamin C. When added to the basic medium for plant tissue culture, ascorbic acid inhibits the action of phenolic compounds generated in the culture, thereby reducing browning or blackening of the cultured plant and healthy culture. It has the effect of increasing the production efficiency of seedlings.

Ascorbic acid added to the antioxidant medium is vitamin C. When added to the basic medium for plant tissue culture, ascorbic acid inhibits the generation of phenolic compounds generated in the culture medium and causes browning or blackening of the cultured plant. It has the effect of reducing the phenomenon and increasing the production efficiency of healthy cultured seedlings. Glycine added to the medium is one of the amino acids, and serves as an energy source to supply reduced nitrogen such as ammonium ions to cultured plants, and myoinositol is the composition of the vitamin B complex. It is an inositol-type component, which is a component of the body's membrane, and is used for cell wall biosynthesis or polysaccharide biosynthesis.It is used as a growth promoting material in plant tissue culture, and is used not only in plant callus culture, but also in culture and growth point culture. have.

In a specific embodiment of the present invention, the present inventors used CuSO4·5H ₂ O 0.025∼0.2mg/L, Myo-Inositol 0.1 in the same medium condition in order to confirm the antioxidant treatment conditions optimized for plant shoot induction from the growth point of apple dwarf stocks. ∼0.8g/L, 0.5∼4mg/L of Glycine, and 0.0125∼0.1g/L of Casein hydrolysate were added and compared. When treated with 4mg/L of Glycine, both M.9 stocks and M.26 stocks had low browning rates. And a high survival rate.

Carbon sources are needed to supply carbohydrates to the cultured tissue. Various types of saccharides added to MS media are essential for tissue culture media because they act as energy sources for cultured tissues that do not have the ability to synthesize carbohydrates. The types of sugars used in tissue culture include sucrose, glucose, fructose, maltose, etc. Among them, sucrose is a monosaccharide in the process of dissolving in water and autoclaving. It is decomposed into phosphorus glucose and fructose, and is released from the cell membrane of the culture medium, and is hydrolyzed by invertase (an enzyme that hydrolyzes sucrose, a disaccharide into monosaccharides, fructose and glucose) or extracellular enzyme. It is most widely used because the culture changes to a form that is easy to absorb as it is decomposed.

In a specific embodiment of the present invention, in order to confirm the carbon source treatment conditions optimized for the induction of plant shoots from the growth point of the apple dwarf stock, the present inventors use sucrose 10 to 70 g/L for each carbon source type, glucose 10 to 70 g/L, and As a result of comparison by adding 10 to 70 g/L of lactose and 10 to 70 g/L of sorbitol for each concentration, both M.9 stocks and M.26 stocks had the best growth characteristics of in-flight plants under the condition of glucose 30 g/L. Was confirmed.

In the disease-free production method of apple dwarf stocks of the present invention, the second MS medium is in MS medium at a concentration of 4 to 5 g/L, BAP 0.8 to 1.2 mg/L, IBA 0.1 to 0.5 mg/L, glycine 2 to 6 mg/L, glucose may contain 25 to 35 g/L. Preferably, it may contain 1.0 mg/L of BAP, 0.2 mg/L of IBA, 4 mg/L of glycine, and 30 g/L of glucose in MS medium at a concentration of 4.40 to 4.42 g/L.

In the method for producing bottle-free apple dwarf stocks of the present invention, the third MS medium may contain 0.3 to 0.7 mg/L of IBA in the MS medium at a concentration of 2 to 3 g/L. Preferably, 0.5 mg/L of IBA may be included in the MS medium at a concentration of 2.1 to 2.3 g/L.

The third MS medium may further contain 25 to 35 g/L of glucose.

In the method for producing a disease-free apple dwarf stock of the present invention, the first, second, and third MS medium may further contain ascorbic acid, agar, and PPM (Plant Preservative Mixture). .

In a specific embodiment of the present invention, the present inventors collected axillary buds of apple dwarf stocks M.9 and M.26 and cultured in a growth point culture medium to confirm the growth point. It was confirmed that shoot formation and development were confirmed by culturing in a medium for growing shoots with optimized conditions, and it was confirmed that the shoots were cultured in a medium for differentiation of young plants to induce the formation of young plants. In addition, it was confirmed that the roots of the young plants were formed by culturing the young plants in a medium for rooting induction, and it was confirmed that the seedlings were formed by purifying the young plants from which the rooting was induced.

Accordingly, the method for producing a bottle of apple dwarf stocks according to the present invention can be usefully used for in-flight mass propagation of apple dwarf stocks by using a suitable medium and culture conditions for each culture step.

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 by the following examples.

<Example 1> Cultivation of fresh spots from apple dwarf stock sprouts

Axillary buds of 5-year-old M.9 and M.26 apples planted on the test package of the Chungcheongbuk-do Agricultural Technology Institute (Axillary bud) are collected, and the base of the axilla (the part of the root under the axilla attached to the branch stem) ) Was carefully removed so that the woody part of about 5mm was attached to it, and the surface was washed for 2 minutes in running water in a laboratory. Disinfect the surface of the liquid by immersing it in a sterilized glass bottle (81 mm outer diameter, 132 mm height) containing 150 ml of 70% ethanol in a clean bench (sterile workbench) for 30 seconds and sterilize with 150 ml of 3% sodium hypochlorite (NaOCl) solution It was immersed in a glass bottle for 15 minutes, sterilized once, and then taken out and washed three times for 5 minutes each in a glass bottle containing 150 ml of sterilized distilled water. After that, in order to aseptically collect the growth points of the liquid buds that have been cleaned and disinfected, use a stereo microscope (Stemi 2000, Carl Zeiss) in a clean bench (aseptic workbench) to determine the growth point (0.1~0.3mm) in the liquid buds as a leaf primordium. A group of cells that become the root of the leaf or generate the original leaf) was collected in a state attached to it, and plated on MS basic medium containing the components of Table 1 and cultured.

Last name content NH ₄ NO ₃ 1650.00 mg/L MgSO ₄ 180.54 mg/L KNO ₃ 1900.00 mg/L KH ₂ PO ₄ 170.00 mg/L CaCl ₂ 332.02 mg/L CoCl ₂ 6H ₂ O 0.025 mg/L ZnSO ₄ 7H ₂ O 8.60 mg/L Na ₂ MoO ₄ 2H ₂ O 0.25 mg/L MnSO ₄ H ₂ O 16.90 mg/L H ₃ BO ₃ 6.20 mg/L KI 0.83 mg/L FeNaEDTA 36.70 mg/L CuSO ₄ 5H ₂ O 0.025 mg/L Glycine 2.00 mg/L Myo-Inositol 100.00 mg/L Thiamine HCl 0.1 mg/L Nicotinic acid 0.5 mg/L Pyridoxine HCl 0.5 mg/L

Initial growth point Each growth point was placed in a culture glass test tube (diameter 25㎜, height 150㎜) containing the culture medium, and in bright condition (23±2℃, 16/8h light/dark photoperiod, 40 μmol·m ^-2 ) Incubated for 2-3 weeks. In the case of browning or blackening in which the medium or growth point culture changes to brown or black during the culture period for 2-3 weeks, the growth point was immediately transferred to a new medium and cultured for 6 weeks.

<Experimental Example 1> Establishment of optimal culture conditions for formation of shoots from the growth point

1-1. Check basic badge processing conditions

The composition of the basic medium in the early stage of growth point culture is a medium that is a balanced mixture of inorganic salts, vitamins, and energy sources. MS (Murashige & Skoog, 1962) medium, which is widely used as a basic medium when culturing plant tissues, 2.2∼8.8g/L, wood 1/2 of WPM (McCown and Lloyd, 1981) medium 1.19-4.76g/L, MS medium 1.1-4.4g/L, and WPM medium 0.595-2.38g/L developed for cultivation of trees or shrubs as a medium for use , 1, 2 times the concentration was treated. Each medium contains 0.1 g/L of ascorbic acid and 30 g/L of sucrose (Junsei, Tokyo, Japan). 9 g/L of agar (Junsei Chemical, Tokyo, Japan) was added, and then mixed and prepared, and the pH of the medium was adjusted to 5.8. Then, 10 to 15 ml each was dispensed into a culture glass test tube (diameter 25 mm, height 150 mm), and autoclaved (121°C, 1.2 kgf· ^-2 pressure, 15 minutes). Culture conditions were incubated for 4 weeks in bright culture (light cycle 16D: 8H, cool white fluorescent lamp 40 ㎛ol·m ^-2·s-1 ) in a culture room maintained at 23±1℃ for 4 weeks, and the number of shoots, shoot length, and survival rate The growth characteristics of young plants were investigated by comparing them.

Medium composition M.9 MS WPM New chosu New chapter Survival rate (g/L) (g/L) (recast) (Cm) (%) 2.2 - 1.7 ab ^z 4.3 bc 100.0 a 4.4 - 2.3 a 6.3 a 100.0 a 8.8 - 1.6 bc 4.9 ab 91.3 b - 1.19 1.1 c 3.1 c 97.5 a - 2.38 1.6 ab 3.7 bc 98.9 a - 4.76 1.3 abc 4.5 bc 100.0 a 1.1 0.59 1.6 ab 3.9 bc 98.3 a 2.2 1.19 1.8 ab 5.1 ab 100.0 a 4.4 2.38 1.7 ab 4.9 ab 98.3 a

^z DMRT 5%

Medium composition M.26 MS WPM New chosu New chapter Survival rate (g/L) (g/L) (recast) (Cm) (%) 2.2 - 2.0 ab 4.3 d 100.0 a 4.4 - 2.2 a 6.9 a 100.0 a 8.8 - 1.9 b 6.8 ab 100.0 a - 1.19 1.0 c 5.2 cd 85.0 a - 2.38 1.0 c 5.3 cd 100.0 a - 4.76 1.0 c 4.3 d 100.0 a 1.1 0.59 1.0 c 5.9 bc 100.0 a 2.2 1.19 1.3 c 4.6 cd 100.0 a 4.4 2.38 1.0 c 4.2 d 100.0 a

^z DMRT 5%

As a result, as shown in Tables 2 and 3, when MS medium was treated than WPM medium, the number of shoots was 1.5 to 2 times higher, and M.9 stocks were better than M.26 stocks. , M.9 and M.26 stocks were treated with MS 4.4g/L, it was confirmed that the number of shoots was 2.3/week, the shoot length was 6.3cm, and the survival rate was 100%.

1-2. Confirmation of carbon source treatment conditions

Various types of saccharides added to MS media are essential for tissue culture media because they act as energy sources for cultured tissues that do not have the ability to synthesize carbohydrates. Types of carbon sources include sucrose, glucose, fructose, and maltose, and to confirm the optimal carbon source treatment conditions for the formation of shoots from the growth point of apple dwarf stocks, MS (Murashige & Skoog, 1962) medium 4.4g/L, agar 9g/L , Ascorbic acid 0.1g/L, PPM(Plant Preservative Mixture, Plant Cell Technology) 2.0ml/L, sucrose 10~70g/L, glucose 10~70g/L, fructose 10~70g /L and sorbitol 10 to 70g/L were added for each concentration to prepare a mixture, and then the pH of the medium was adjusted to 5.8. Then, 100 ml each was dispensed into a plant culture vessel (outer diameter 65 mm, height 130 mm), and autoclaved (121° C., 1.2kgf·cm ^-2 pressure, 15 minutes). Young plants were placed in 2-3 culture bottles and cultured for 4 weeks in a bright state (23±1℃, 16/8h light and dark cycle, 40μmol·m ^-2 ). The number of shoots, shoot length, number of leaves, and survival rate were compared. The growth characteristics of plants were investigated.

process M.9 Carbon source
(g/L) New chosu
(recast) New chapter
(Cm) ground game
(recast) Survival rate
(%) Sucrose 10 1.5 df ^z 2.1 e 8.6 cd 88.3 a Sucrose 30 2.2 a-d 2.5 de 10.5 b-d 98.4 a Sucrose 50 1.6 c-f 2.7 c-e 7.9 d 100.0 a Sucrose 70 2.2 a-d 3.3 c 13.8 a-c 100.0 a Glucose 10 2.4 ab 3.3 c 6.6 d 75.0 a Glucose 30 2.6 a 4.9 a 15.8 a 100 .0 a Glucose 50 2.4 ab 4.2 b 14.3 a 100.0 a Glucose 70 1.3 ef 4.0 a 8.9 cd 100.0 a Fructose 10 1.5 ef 4.2 b 8.3 d 86.7 a Fructose 30 1.0 f 2.7 c-e 5.6 d 88.3 a Fructose 50 1.8 b-e 2.7 c-e 10.5 b-d 95.0 a Fructose 70 1.3 ef 2.6 c-e 6.9 d 91.7 a Sorbitol 10 2.2 a-d 2.9 cd 15.2 a 96.7 a Sorbitol 30 2.4 ab 3.3 c 13 ab 90.0 a Sorbitol 50 1.6 c-f 2.3 de 9.8 b-d 83.4 a Sorbitol 70 1.1 ef 2.1 e 5.7 d 50.0 b

^z DMRT 5%

process M.26 Carbon source
(g/L) New chosu
(recast) New chapter
(Cm) ground game
(recast)) Survival rate
(%) Sucrose 10 1.7 eg ^z 3.8 c-e 14.3 b-e 96.5 a Sucrose 30 2.4 ab 5.6 ab 14.7 a-d 100.0 a Sucrose 50 2.0 b-e 5.2 a-c 14.3 b-e 100.0 a Sucrose 70 2.2 a-d 5.1 a-c 13.9 c-e 100.0 a Glucose 10 2.2 a-d 4.9 b-d 12.3 c-e 98.4 a Glucose 30 2.5 a 5.9 a 18.1 a 100.0 a Glucose 50 2.3 a-c 5.9 a 15.6 ab 98.4 a Glucose 70 2.2 a-d 5.2 a-c 10.7 ef 95.0 a Fructose 10 1.9 d-g 5.2 a-c 11.8 de 95.0 a Fructose 30 1.1 hi 3.3 d-f 4.0 g 65.0 b Fructose 50 1.5 gh 4.2 b-d 7.6 f 88.4 ab Fructose 70 1.0 i 3.3 d-f 3.8 g 64.9 b Sorbitol 10 2.1 b-e 4.2 b-d 13.1 c-e 100.0 a Sorbitol 30 2.3 a-c 3.3 d-f 17.7 ab 98.3 a Sorbitol 50 2.4 ab 2.4 ef 17.1 ab 98.4 a Sorbitol 70 1.6 fg 1.7 f 7.6 f 76.7 a

^z DMRT 5%

As a result, as shown in Tables 4 and 5,

The overall growth characteristics were good when treated with carbon source sucrose and glucose, and M.9 stocks showed 2.6 shoots/week, leaves 15.8/week, and 100% survival rate at a glucose concentration of 30g/L, and M.26 stocks showed 30g glucose. At /L concentration, it was confirmed that the growth characteristics of in-flight plants were best under the condition of glucose 30g/L concentration with 2.5 shoots/week, 18.1 leaves/week, and a survival rate of 100%.

1-3. Confirmation of plant growth regulator treatment conditions

Plant growth regulators are a generic term for synthesized hormonal chemicals that have a great effect on plant growth even in small amounts, and are cultured for 6 weeks to identify optimal plant growth regulators for shoot formation from the growth point of apple dwarf stocks. One plant was cultured for 4 weeks for each type of plant growth regulator. Plant growth regulators, auxin and cytokinin, play an important role in the in-flight culture of higher plants every week, and their addition method and concentration for elongation and division of cultured cells depend on the type and type of culture. Do it differently. The formation of roots and shoots is due to the interaction of auxin and cytokinin, and in order to form them well, the ratio of these two plant growth regulators must be appropriate. Plant growth regulator medium conditions are MS (Murashige & Skoog, 1962) medium 4.4g/L glucose 30g/L, agar 9g/L, ascorbic acid 0.1g/L, PPM (Plant Preservative Mixture, Plant Cell) Technology) 2.0ml/L BAP 1mg/L IBA 0.2mg/L, kinetin 0.5mg/L IBA 0.2mg/L, GA3 0.5mg/L IBA 0.2mg/L, TDZ 0.5mg/L IBA 0.2mg/L of IBA at 0.2mg/L and 2mg/L of BAP, 0.2mg/L of kinetin 0.5mg/L of IBA, 0.2mg/L of GA3 0.5mg/L of IBA, 0.2mg/L of TDZ 0.5mg/L of IBA After mixing and preparing each L was added, the pH of the medium was adjusted to 5.8. Then, 100 ml each was dispensed into a plant culture vessel (outer diameter 65 mm, height 130 mm), and autoclaved (121°C, 1.2kgf·cm ^-2 pressure, 15 minutes) and used. The young plants were placed in 2-3 culture bottles and cultured for 4 weeks in the bright state (23±2℃, 16/8h light and dark cycle, 40μmol·m ^-2 ). The number of shoots, shoot length, and survival rate were compared. Growth characteristics were investigated.

Plant growth regulator M.9 M.26 BAP
(mg/L) IBA
(mg/L) PGRs
(mg/L) New chapter
(Cm) New chosu
(recast) Survival rate
(%) New chapter
(Cm) New chosu
(recast) Survival rate
(%)

One - - 6.5 a ^z 2.1 ab 92.1 ab 8.3 a 2.5 ab 90.0 b 0.2 - 6.4 a 2.3 a 97.4 a 8.2 a 2.6 a 100.0 a 0.2 Kinetin 0.5 4.5 ab 2.3 a 93.4 ab 5.8 a 2.1 b~e 97.5 a 0.2 GA3 0.5 5.9 ab 1.9 ab 87.1 a~c 5.9 a 2.3 a~d 100.0 a 0.2 TDZ 0.5 4.0 b 1.4 c 86.1 a~c 5.8 a 1.8 ef 100.0 a


2 - - 4.7 ab 2.2 a 65.8 cd 5.8 a 2.4 a~c 100.0 a 0.2 - 3.9 b 1.7 bc 80.6 a~c 5.3 a 1.9 de 100.0 a 0.2 Kinetin 0.5 4.9 ab 2.1 ab 79.2 a~c 6.9 a 2.4 a~c 100.0 a 0.2 GA3 0.5 3.1 b 1.4 c 51.2 d 4.8 a 2.0 c~e 100.0 a 0.2 TDZ 0.5 2.9 b 1.3 c 69.6 b~d 3.9 a 1.4 f 90.0 b

^z DMRT 5%

As a result, as shown in Table 6,

Plant growth regulators auxin and cytokinin were mixed and treated with auxin-type BAP 1mg/L and auxin-type IBA 0.2mg/L treatment. I confirmed the best. Skoog and Miller (1957) found that when the concentration of cytokinin is higher than that of auxin when cultivating tobacco water (pith) tissue, the formation of shoots is promoted, and when the vice versa, the formation of roots is promoted. Reported that the formation of

1-4. Confirmation of antioxidant treatment conditions

In woody plants, when tissue is cultured, phenolic compounds escape into the medium, browning the medium, causing a decrease in the survival rate.Therefore, there is a correlation between the level of phenolic substances in the tissue and the death rate of the culture. To do this, an experiment was conducted by treating antioxidants by type. MS (Murashige & Skoog, 1962) medium 4.4g/L glucose 30g/L, agar 9g/L, Ascorbic acid 0.1g/L, PPM (Plant Preservative Mixture, Plant Cell Technology) 2.0ml/L After adding CuSO4·5H ₂ O 0.025∼0.2mg/L, Myo-Inositol 0.1∼0.8g/L, Glycine 0.5∼4mg/L, Casein hydrolysate 0.0125∼0.1g/L were added and mixed. The pH was adjusted to 5.8. Then, 100 ml each was dispensed into a plant culture vessel (outer diameter 65 mm, height 130 mm), and autoclaved (121° C., 1.2kgf·cm ^-2 pressure, 15 minutes). The young plants were placed in 2-3 culture bottles and cultured for 4 weeks in the bright state (23±1℃, 16/8h light and dark cycle, 40μmol·m ^-2 ), and the number of shoots, shoot length, browning rate, and survival rate were compared. Growth characteristics of young plants were investigated.

process M.9 M.26 Antioxidant New chosu New chapter Browning rate Survival rate New chosu New chapter Browning rate Survival rate (recast) (Cm) (%) (%) (recast) (Cm) (%) (%) Control 2.8 b ^z 4.8 c 43.5 a 70.3 c 2.8 b 5.0 c 37.0 a 74.6 c CuSO ₄ ㆍ5H ₂ O 0.025 mg/L 2.4 c 5.7 b 25.9 bc 84.5 b 2.5 c 5.4 bc 26.2 bc 83.4 b CuSO ₄ ㆍ5H ₂ O 0.05 mg/L 2.7 bc 5.5 b 25.1 bc 82.3 b 2.0 c 5.6 b 22.7 bc 82.9 b CuSO ₄ ㆍ5H ₂ O 0.1 mg/L 2.9 b 5.3 bc 27.1 bc 84.8 b 2.6 bc 5.5 bc 25.1 bc 80.0 bc CuSO ₄ ㆍ5H ₂ O 0.2 mg/L 3.0 b 5.8 b 20.7 c 87.8 ab 2.9 b 5.7 b 18.5 c 85.2 b Myo-Inositol 0.1 g/L 2.9 b 5.3 bc 29.6 bc 84.2 b 2.6 bc 5.9 b 27.5 bc 83.4 b Myo-Inositol 0.2 g/L 3.4 a 6.2 ab 22.4 c 86.1 ab 3.6 a 6.3 b 20.7 c 84.3 b Myo-Inositol 0.4 g/L 2.9 b 6.0 ab 24.6 bc 85.2 b 3.1 ab 5.9 b 21.7 c 82.7 bc Myo-Inositol 0.8 g/L 2.5 c 6.1 ab 26.6 bc 83.0 b 2.6 bc 6.1 b 28.3 bc 82.7 b Glycine 0.5 mg/L 2.7 bc 6.1 ab 11.6 d 86.5 ab 2.9 b 5.8 b 11.2 d 85.0 b Glycine 1 mg/L 2.8 bc 6.2 ab 10.8 d 88.5 a 3.2 a 5.8 b 15.5 cd 87.0 ab Glycine 2 mg/L 3.3 ab 6.4 a 11.6 d 91.7 a 3.4 a 6.1 b 10.7 d 86.3 ab Glycine 4 mg/L 3.5 a 6.5 a 10.6 d 94.0 a 3.7 a 6.5 a 8.4 d 89.5 a Casein hydrolysate 0.0125 g/L 2.4 c 4.9 b 37.1 ab 80.1 bc 3.1 ab 5.3 bc 34.7 ab 78.0 bc Casein hydrolysate 0.025 g/L 3.0 b 5.0 c 30.5 b 81.4 b 3.2 ab 5.4 bc 31.4 b 80.6 bc Casein hydrolysate 0.05 g/L 2.8 bc 4.8 c 33.7 ab 83.4 b 2.9 b 5.3 bc 32.7 ab 75.7 c Casein hydrolysate 0.1 g/L 2.4 c 4.6 c 34.6 ab 79.3 bc 2.8 bc 4.9 c 32.8 ab 65.7 d

^z DMRT 5%

As a result, as shown in Table 7,

When 4 mg/L of glycine, an antioxidant, was added, the browning rate of M.9 stock was 4 times lower at 10.6% compared to 43.5% without treatment, and the survival rate was the highest at 94%. In the case of M.26 stocks, the browning rate was 8.4% lower than that of untreated 37.0%, and the survival rate was also the highest at 89.5%.

<Example 2> Induction of differentiation of young plants

After going through the cultivation process that induces the growth of shoots formed from the growth point, a cultivation step is required to elongate shoots and speed up the growth rate to differentiate into young plants (powders). Two to three shoots were placed in a culture vial (Plant culture vessle, outer diameter 65 mm, height 130 mm) containing the young plant culture medium containing the ingredients in Table 1, and in bright condition (23±2℃, 16 /8h light/dark photoperiod, 40 μmol·m ^-2 ) for 4 weeks. Examples are ascorbic acid 0.1 g/L, BAP 1 mg/L and IBA 0.2 mg/L, glycine 4 mg/L, glucose 30 g/L, agar 9 g in 4.4 g/L of MS medium /L, PPM (Plant Preservative Mixture, Plant Cell Technology) 2.0 ml/L was mixed and prepared, and then the pH of the medium was adjusted to 5.8. The control was cultured in a medium to which only the same concentration of PPM, sucrose, and Agar was added to the same basic MS medium as in the Example. The young plants were placed in 2-3 culture bottles and cultured for 8 weeks in a bright state (23±2℃, 16/8h light and dark cycle, 40μmol·m ^-2 ). The number of shoots, shoot length, number of leaves, and survival rate were checked. The growth characteristics of plants were investigated.

division M.9 M.26 New chosu New chapter Survival rate New chosu New chapter Survival rate (recast) (Cm) (%) (recast) (Cm) (%) Example 4.4 6.1 100.0 4.7 6.7 100.0 Control 2.8 4.8 70.3 2.8 5.0 74.6

As a result, as shown in Table 8, in the case of M.9, the number of shoots was 4.4 per week, the height of shoots was 6.1 cm, and the survival rate was 100%, showing good growth characteristics in the examples, and in the case of M.26, the number of shoots was 4.7 pieces/week. It was confirmed that the growth characteristics were better in the examples than the control with the main, new height of 6.7cm and survival rate of 100%.

<Example 3> Induction of rooting of young plants

It was cultured using the prepared medium to form the roots of the young plants cultured in the young plant medium for 4 weeks. Rooting medium is Murashige & Skoog Medium (MS medium, M0245, Duchefa) 2.2g/L, glucose 30g/L, ascorbic acid 0.1 g/L, agar 9g/L, PPM (Plant Preservative Mixture, Plant Cell Technology) IBA 0.5 mg/L, IBA 1 mg/L, IBA 0.5 mg/L each in 1 ml/L Paclobutrazol 1 mg/L, IBA 0.5 mg/L Paclobutrazol 1.5 mg/L, IBA 0.5 mg/L Myo-inositol 0.2mg/L, IBA 0.5mg/L· Myo-inositol 0.4mg/L were respectively treated to prepare a mixture, and then the pH of the medium was adjusted to 5.8. Then, 100 ml each was dispensed into a plant culture vessel (outer diameter 65 mm, height 130 mm), and autoclaved (121°C, 1.2 kgf·cm ^-2 pressure, 15 minutes). Two to three young plants were placed in a culture vial (Plant culture vessle, outer diameter 65 mm, height 130 mm) containing seedling plant rooting medium, and in bright state (23±2℃, 16/8h light/dark photoperiod) , 40 μmol·m ^-2 ) for 8 weeks.

process M.9 IBA
(mg/L) PGRs
(mg/L) New chapter ground game Root number Root length Survival rate (cm) (recast) (recast) (mm) (%) - - 2.4 c ^z 5.1 c 1.3 ab 13.8 bc 39.0 c 0.5 - 3.8 a 11.7 a 2.3 a 32.1 a 100.0 a 1.0 - 2.9 b 10.5 ab 1.4 ab 21.0 abc 73.3 a 0.5 Paclobutrazol 1.0 2.3 c 7.4 b 0.6 b 10.5 bc 80.0 ab 0.5 Paclobutrazol 1.5 1.9 c 7.2 b 0.4 b 5.9 c 53.3 c 0.5 Myo-Inositol 0.2 3.1 b 10.6 ab 1.6 ab 28.7 ab 100.0 a 0.5 Myo-Inositol 0.4 3.2 b 10.5 ab 1.2 ab 21.1 abc 66.6 bc

^z DMRT 5%

process M.26 IBA
(mg/L) PGRs
(mg/L) New chapter ground game Root number Root length Survival rate (Tsu) (recast) (recast) (mm) (%) - - 2.7b ^z 5.3c 0.6a 8.4b 86.0b 0.5 - 3.9a 10.3a 1.7a 26.5a 100.0a 1.0 - 3.8a 9.9a 1.3a 39.2a 90.0a 0.5 Paclobutrazol 1.0 2.5b 6.1a 1.5a 25.7a 86.7b 0.5 Paclobutrazol 1.5 2.3b 6.5b 0.9a 28.5a 83.3b 0.5 Myo-Inositol 0.2 3.8a 8.7ab 0.8a 36.3a 100.0a 0.5 Myo-Inositol 0.4 3.9a 9.9a 0.7a 38.2a 100.0a

^z DMRT 5%

As a result, as shown in Tables 9 and 10,

In the rooting induction stage, when the MS (Murashige & Skoog, 1962) medium 2.2g/L and IBA 0.5 mg/L is included in the medium, shoot length 3.8~3.9cm, leaves 10.3~11.7/week, root number 1.7~2.3 /Week, it can be seen that the survival rate of 100% can induce the best rooting.

<Example 4> Out-of-air purification

, 16/8h light/dark photoperiod, 40~50 μmolㆍm^-2, 공중습도 60±5%)의 순화실에서 1~2주 키운 다음, 최고 25℃ 최저 18℃로 유지되는 유리 온실 또는 비닐하우스에서 공중습도를 서서히 낮춰주면서 1~2주 정도 경화시켜 가면서 키웠다. 기외 순화 4주 후부터는 유식물체의 상태를 확인하면서 공중습도를 천천히 낮춰주면서 외기의 환경에 노출시켜 완전히 경화시켰다.Since the roots are completely formed, a young plant being cultured in a culture vial can be taken out of the culture vessel and subjected to an external purification process in which it adapts to the external environment to obtain a complete plant shape. Therefore, to purify outside the air, take out the young plants of the apple dwarf stocks M.9 and M.26 with roots from the culture glass bottle, and gently wash them with water so that the culture medium does not remain on the roots of the young plants, then pearlite: vermiculite. (Vermic stone) Transplanted into a pot (internal diameter 110 mm, height 113 mm) containing artificial soil mixed in 1:1 (v:v). Potted young plants are in an environment similar to a culture room (23±2

, 16/8h light/dark photoperiod, 40~50 μmolㆍm ^-2 , air humidity 60±5%) in a purifying room for 1-2 weeks and then maintained at a maximum of 25℃ and a minimum of 18℃ in a glass greenhouse or green house It was raised by gradually lowering the air humidity and curing for 1 to 2 weeks. After 4 weeks of acclimatization outside the air, while checking the condition of the plants, slowly lowering the air humidity and exposing it to the outside air to completely cure.

All the treatments in the above examples were measured repeatedly three times in a completely random batch, and the analysis of the test results was performed using an analysis of variance (ANOVA) using COSTAT (CoHort software, Berkeley, CA, USA), a statistical package for PC, and then Duncan' Multiple Range The significance was tested at p <0.05 level with Test (DMRT).

Claims (10)

The growth point was collected from Axillary buds of M.9 or M.26, a dwarf apple cultivar, and the collected growth point was BAP (6-benzylamino purine) 0.8 to 1.2 mg/L, IBA (indole-3-butyric acid). ) 0.1 to 0.5 mg / L, glycine (Glycine) 2 to 6 mg / L and glucose (Glucose) 25 to 35 g / L containing the first MS (Murashige and Skoog basal) medium containing the step of growing shoots ;
Culturing the grown shoots in a second MS medium containing BAP, IBA, glycine and glucose to differentiate into young plants;
Inducing rooting by culturing the young plant in a third MS medium containing IBA (Indole-3-Butyric Acid); And
Purifying the young plant from which the rooting has been induced; Apple dwarf stock production method comprising a.
delete The method of claim 1,
The first MS medium,
In MS medium at a concentration of 4.40 to 4.42 g/L,
BAP 1.0 mg / L, IBA 0.2 mg / L, glycine 4 mg / L, characterized in that the medium prepared by adding glucose 30 g / L, apple dwarf stock production method.
The method of claim 1,
The second MS medium,
In MS medium at a concentration of 4 to 5 g/L,
BAP 0.8 to 1.2 mg / L, IBA 0.1 to 0.5 mg / L, glycine 2 to 6 mg / L, characterized in that the medium prepared by adding glucose 25 to 35 g / L, apple dwarf stock production method.
The method of claim 1,
The second MS medium,
In MS medium at a concentration of 4.40 to 4.42 g/L,
BAP 1.0 mg / L, IBA 0.2 mg / L, glycine 4 mg / L, characterized in that the medium prepared by adding glucose 30 g / L, apple dwarf stock production method.
The method of claim 1,
The third MS medium,
A method for producing apple dwarf stocks, characterized in that it is a medium prepared by adding 0.3 to 0.7 mg/L of IBA to MS medium of 2 to 3 g/L concentration.
The method of claim 1,
The third MS medium,
A method for producing apple dwarf stocks, characterized in that it is a medium prepared by adding 0.5 mg/L of IBA to MS medium at a concentration of 2.1 to 2.3 g/L.
The method according to claim 6 or 7,
The third MS medium is characterized in that it further contains 25 to 35 g / L of glucose, apple dwarf stock production method.
The method of claim 1,
The first, second and third MS medium, characterized in that it further comprises ascorbic acid (Ascorbic acid), agar (agar) and PPM (Plant Preservative Mixture), apple dwarf stock production method.
The method of claim 1,
The pH of the first MS medium and the second MS medium is 5 to 6, characterized in that the apple dwarf stock production method.

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