KR102200935B1 - Method for plant formation of jujube 'Bokjo' cultivar(Ziziphus jujuba Mill) using internode culture - Google Patents

Abstract

The present invention comprises the steps of: 1) inducing adventitious embryos by culturing the internodes of'demonstration', which is a variety of Jujube trees (Ziziphus jujuba Mill), in a medium for inducing adventitious embryos; 2) culturing the adventitious embryo of step 1) in a shoot growth medium to form shoots and culturing them; 3) culturing the shoots of step 2) in a culture medium for differentiation of young plants to differentiate into young plants; 4) inducing rooting by culturing the young plant of step 3) in a medium for rooting induction; And 5) producing an adult tree by purifying the young plant from which the rooting is induced in step 4) out of the air; to a method for forming a plant for mass proliferation of a jujube tree'demodulation' comprising, the plant forming method of the present invention By using a suitable medium and culture conditions for each cultivation step, the plant of the present invention is superior to the case of culturing in a basic medium, so that the adventitious bud formation rate, shoot formation rate and shoot growth, seedling plant formation rate, rooting rate and growth of adult trees are superior. The formation method can be usefully used in mass production of'demolition' of jujube trees.

Description

Method for plant formation of jujube'Bokjo' cultivar(Ziziphus jujuba Mill) using internode culture}

The present invention relates to a method for forming a plant for mass propagation of a jujube tree'demodulation'.

The jujube tree ( Ziziphus jujuba Mill) is one of the deciduous trees belonging to the Rhamnaceae family, and its origin is known as China, and the age that was introduced to Korea has not been clearly known, but has been widely cultivated since 1188 (18 years of Myeongjong). . Jujube trees are distributed and vegetated not only in Korea, but also in China, Japan, and southern Europe. The best cultivation areas have an annual average temperature of 8℃ or higher, an average temperature of -10℃ or higher in January, and an annual minimum temperature of -28℃ or higher. good. Leaves are ovate, pointed at the end, rounded at the bottom, not the same on the left and right sides, blunt serrated edges and glossy. The flowers are yellow-green, bloom in May-June, and two or three small flowers hang on a bisexual flower, a flower blooms at the end of the flower stalk, and branches are split again from there. The fruit is a drupe (the fruit surrounded by a hard core with seeds), spherical or oval, and ripens in reddish brown or dark brown from September to October. The fruit of the jujube tree is not only raw, but also dried after harvesting and used as a dried fruit for snacks, cooking and medicinal purposes. It is also used as jujube liquor, jujube tea, jujube vinegar, and jujube porridge, and honey jujube as a processed product is well received in China, Japan and Europe. The fruit of the jujube tree is rich in inorganic components, amino acids, vitamin C, dietary fiber, and beta-carotene, and is known to have anti-aging, anti-cancer effects, blood circulation, etc., and is also used as a diuretic, tonic, and emollient in herbal medicine. have.

Due to the spread of well-being culture, domestic jujube production (production value) is increasing every year, which is 14,236 tons (99.1 billion won) in 2014, an increase of 50% compared to 2010. Jujube, mainly cultivated in Korea, is a cultivar of'decomposition', and in the past, it has been mainly used as a herbal ingredient in the form of dried fruits (Eom et al., 2016; Oh et al., 1988). It is in the spotlight as a new high-income crop as it is mainly sold raw jujube in Boeun, North Chungcheong Province. According to statistics from the Korea Forest Service (KFS, 2016), the national jujube cultivation area in 2015 was 2,744 ha. It was 11.3%, but in terms of production, Gyeongsan 32.2%, Boeun 25.5%, Gunwi 13.4%, Qingdao 10.6%, Miryang 8.4%, and other regions 9.9%.

Conventional jujube tree seedling propagation, seedling, root suction (吸枝, a thing that grows from an underground stem and then appears on the ground and becomes an independent individual after being separated from the parent body), grafting Or, a method of producing grafted seedlings by attaching to a stem with snow (reception) and a stem or root with roots (large tree), cuttings (揷木, a part of the plant's nutritional organs, separated from the parent body and in soil or sand) Methods such as plugging and rooting, germinating and breeding as independent plants) have low growth rates and are non-uniform. Therefore, in order to stably produce and distribute high-quality and superior jujube tree seedlings to Korea, it is necessary to develop efficient jujube tree seedling production technology that can improve the problems of conventional breeding methods. Therefore, the development of high-quality, disease-free mass propagation technology to establish the basis for stable production of high-quality jujube is more demanded than ever. In foreign countries, the use of micropropagation technology that can mass-produce superior and uniform seedlings at once is universal and is in the stage of practical use.

The technology to produce intact plants by culturing tissues such as eyes, leaves, stems, and roots of plants in an artificial in-flight environment is called "tissue cultivation". In a culture chamber where temperature and humidity are controlled, culture containers such as glass bottles or test tubes It is a technology that puts nutrients in which plants can grow and grows plants in a sterile condition 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 intrinsic differentiation ability of plants is called totipotency, which 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. . In particular, various nutrients are required to grow and proliferate in a special environment for plant tissues or organs to grow and proliferate in a special environment such as in-flight (cultivation test tubes or glass bottles), but the requirement of nutrients depends on the type of plant or the type of cultured tissue or organ. Since there are differences, 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 the "demodulated" variety for mass propagation and production of young plants of jujube tree "demonstration".

Accordingly, the inventors of the present invention cultivate the internode (between nodes and nodes on which leaves are attached to the stem of the plant) of the "demonstration" cultivar of jujube tree in the cabin, and directly from them, It is common for plants to have eyes in certain areas, such as leaves, roots, trunks, etc.), and sprout formation and young shoots by germinating adventitious buds. The present invention was completed by establishing a method for efficiently proliferating seedlings of "demodulated" cultivar through an extracorporeal purification process that induces the differentiation of plants (plants) and adapts them to the external environment.

It is an object of the present invention to provide a method for forming a plant for mass propagation of a jujube tree'demodulation'.

In order to achieve the above object, the present invention comprises the steps of: 1) inducing adventitious embryos by culturing the nodes of'demonstration', which is a variety of Ziziphus jujuba Mill, in a medium for inducing adventitious embryos; 2) inducing shoot formation by culturing the adventitious embryo of step 1) in a shoot growth medium; 3) culturing the shoots of step 2) in a culture medium for differentiation of young plants to differentiate into young plants; 4) inducing rooting by culturing the young plant of step 3) in a medium for rooting induction; And 5) purifying the young plant from which the rooting is induced in step 4) out of the air; it provides a method of forming a plant for mass propagation of the jujube tree'demodulation' comprising.

The method of forming a plant for mass proliferation of the jujube tree'demodulation' of the present invention uses a medium and culture conditions suitable for each cultivation step, so that the adventitious bud formation rate, shoot formation rate and shoot growth, young plant Since the formation rate, rooting rate, and growth of adult trees are excellent, the plant formation method of the present invention can be usefully used for mass production of jujube tree'demodulation'.

1 is a diagram showing a shoot and an internode of a jujube tree'demonstration'.
Figure 2 is a diagram showing the adventitious embryo of the jujube tree'demodulation' formed by culturing in the adventitious induction medium of the present invention.
Figure 3 is a diagram showing a shoot formed from adventitious embryos of jujube tree'demonstration' by culturing in the shoot growth medium of the present invention.
4 is a diagram showing a young plant formed from a shoot of a jujube tree'demonstration' by culturing in the culture medium for differentiation of a young plant of the present invention.
Figure 5 is a diagram showing the roots formed from the young plant of the jujube tree'detonation' by culturing in the rooting induction medium of the present invention.
Figure 6 shows a young plant of a jujube tree'demolition' in which rooting is induced and the root is formed . It is a diagram showing the purified trees outside the aircraft.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: 1) inducing adventitious embryos by culturing the internodes of'demonstration', which is a variety of Jujube trees (Ziziphus jujuba Mill), in a medium for inducing adventitious embryos; 2) inducing shoot formation by culturing the adventitious embryo of step 1) in a shoot growth medium; 3) culturing the shoots of step 2) in a culture medium for differentiation of young plants to differentiate into young plants; 4) inducing rooting by culturing the young plant of step 3) in a medium for rooting induction; And 5) purifying the young plant from which the rooting is induced in step 4) out of the air; it provides a method of forming a plant for mass propagation of the jujube tree'demodulation' comprising.

In the plant formation method of the present invention, the medium in each step may be used as a basic medium DKW (Driver and Kuniyuki Walnut) medium.

The DKW medium is a medium in which inorganic salts, vitamins, and energy sources are mixed in a balanced manner, and is a medium widely used as a basic medium for plant tissue cultivation. 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 DKW medium act as energy sources of cultured tissues that do not have the ability to synthesize carbohydrates, and types of sugars used during 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 plant formation method of the present invention, specifically, in each step, the medium uses DKW (Driver and Kuniyuki Walnut) 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 (indolacetic acid), IBA (indole butyric acid, indole butyric acid) and 2,4-D (2,4-dichlorophenoxyacetic acid, 2,4-dichloro). Phenoxyacetic 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-benzylamino purine, 6-benzylaminopurine, 6-BAP), TDZ (thidiazuron) and diphenylurea. (diphenyl urea) 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.

In addition, in the plant formation method of the present invention, specifically, in each step, the medium may further include coconut water, adenine hemisulfate, silver nitrate, PPM, activated carbon, etc., using DKW medium as a basic medium.

Coconut water is a liquid obtained by squeezing the internal flesh of coconut fruit and contains free sugar, amino acids, dietary fiber, vitamins, and plant hormones. It is added to the tissue culture medium of the plant to support the growth of shoots and leaves of the cultured plant. It promotes, increases the size of somatic embryos, and can promote seed germination and regeneration of young plants.

Adenine hemisulfate acts as a biosynthetic precursor of cytokinin among plant hormones, thereby promoting cell division of plants and preventing shoot formation and leaf aging. Therefore, it is added to the tissue culture medium of the plant at an appropriate concentration to promote the action of cytokinin and promote the smooth growth of the cultured plant.

Silver nitrate (AgNO ₃ ) can promote organ differentiation, such as callus formation, embryo development, shoot and root growth, by controlling the growth and development of plants by inhibiting the action of ethylene in plant tissue culture. have. In addition, when the tissue culture of plants such as jujube trees, silver nitrate is added to the culture medium to suppress a sudden browning or black stool of the medium and culture.

Activated charcoal is a powdered charcoal. When added to a plant tissue culture medium, activated charcoal functions to absorb phenolic compounds discharged from in-flight plants or accumulated in the medium. In addition, it promotes the elongation and rooting of shoots, helps to restore the inherent characteristics of plants, and reduces vitrification (anatomical, morphological, and physiological abnormalities of tissue cultured plants) of in-flight cultured plants. can do. However, since it can reduce the growth rate of shoots by absorbing not only the phenolic compounds in the medium but also the growth regulators, it can be added to the medium at an appropriate concentration.

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 plant formation method of the present invention, the medium for inducing adventitious embryos of step 1) is sucrose 10 to 30 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg /L, coconut water 40 to 60 ml/L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.3 to 0.7 Mg/L, TDZ 0.05-0.2 mg/L, IBA 0.05-0.2 mg/L, gibberellin 0.1-0.5 mg/L, and PPM 0.5-1.5 ml/L.

The cultivation of step 1) may be performed for 2 to 3 weeks at a temperature of 20 to 25° C. and an amount of light of 30 to 50 μmol m ^{- 2} .

The medium for growing shoots of step 2) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, coconut water 40 to 60 mL /L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.5 to 1.5 mg/L, TDZ 0.05 to 0.2 mg /L, IBA 0.1 to 0.3 mg/L, Gibberellin 0.1 to 0.5 mg/L, and PPM 0.5 to 1.5 mL/L.

The cultivation of step 2) may be performed for 2 to 3 weeks at a temperature of 20 to 25° C. and an amount of light of 30 to 50 μmol m ^{- 2} .

The medium for differentiation of young plants in step 3) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, coconut water 40 to 60 Ml/L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.5 to 1.5 mg/L, TDZ 0.05 to 0.2 Mg/L, IBA 0.1 to 0.3 mg/L, Gibberellin 0.5 to 0.7 mg/L, and PPM 0.5 to 1.5 mL/L.

The cultivation of step 3) may be performed for 4 to 6 weeks at a temperature of 20 to 25° C. and an amount of light of 30 to 50 μmol m ^{- 2} .

The medium for rooting induction of step 4) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, coconut water 40 to 60 mL. /L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.2 to 0.3 mg/L, IBA 0.5 to 1.5 mg /L and 0.5 to 1.5 ml/L of PPM.

The cultivation of step 4) may be performed for 3 to 5 weeks at a temperature of 20 to 25° C. and an amount of light of 30 to 50 μmol m ^{- 2} .

The step 5) of gioe purifying temperature and 30 to 60 μmol m of from 20 to 25 ℃ ^- and incubated for 1 to 2 weeks in ^two light conditions of, for culturing for 1 to 2 weeks at a temperature of from a temperature 15 to 28 ℃ Can be.

In a specific embodiment of the present invention, the present inventors confirmed the formation of adventitious buds by collecting the internodes of the cultivar of jujube tree'demonstration' and culturing them in a medium for inducing adventitious embryos (see Fig. 2 and Table 2), and It was confirmed that shoot formation and development were confirmed by culturing in a medium (see FIGS. 3 and 4), and it was confirmed that young plant formation was induced by culturing the shoots in a medium for differentiation of young plants (see FIGS. 4 and 6). It was confirmed that the roots of the young plant were formed by culturing the plant in a medium for rooting induction (see Fig. 5 and Table 8), and it was confirmed that the seedling was formed by purifying the young plant from which the rooting was induced (see Fig. 6). ,

Therefore, the method of forming a plant of the present invention can be usefully used in mass production of jujube tree'demodulation' 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> New Induction of adventitious bud formation from the internode

In May, the shoots (bird branches) internodes (between the nodes with leaves and the nodes) of the jujube tree "Bokjo" variety were cut with pruning shears to a length of 1.5 to 2 cm. The collected internode area was immersed in a sterile glass bottle (outer diameter 81㎜, height 132㎜) containing 150 ml of 70% ethanol in a clean bench (sterile workbench) for 30 seconds to disinfect the surface of the internode area, and then 3% sodium hypochlorite ( After sterilizing once by immersing in a sterile glass bottle containing 150 ml of NaOCl) solution for 15 minutes, taken out and washed three times for 5 minutes in a glass bottle containing 150 ml of sterilized distilled water. After that, cut both cut surfaces of the internodes that have been cleaned and disinfected by about 1 mm with a scalpel, and then in the media for inducing adventitious embryos in Table 1 according to the up-and-down direction of the internodes (the apex of the stem of the shoot is initially directed upward). It was ground and cultured. Adventitious embryo induction medium is DKW medium (D0247, Duchefa) 5.6 g / L as shown in Table 1, sucrose (Sucrose, S4842, MBcell) 20 g / L, myo-inositol (myo-Inositol, I4715, MBcell) 0.1 g / L, Ascorbic acid (A4118, MBcell) 0.1 g/L, Citric acid (C4289, MBcell) 0.05 g/L, Coconut water (C5915, Sigma) 50 ml/L, adenine hemisulfate (Adenine hemisulfate, A4557, MBcell) 25 mg/L, Silver nitrate (AgNO ₃ , S4181, MBcell) 1 mg/L, Activated charcoal (C4337, MBcell) 1 g/L, Agar (A5112, MBcell) 9 g/L, BAP (6-Benzylaminopurine, benzylaminopurine, B5812, MBcell) 0.5 mg/L, IBA (Indole-3-butylic acid, indolebutylic acid, I5550, MBcell) 0.1 mg/L, Gibberellic acid , GA ₃ , G5512, MBcell) 0.3 mg/L, TDZ (Thidiazuron, Thidiazuron, T5900, MBcell) 0.1 mg/L, PPM (Plant Preservative Mixture, Plant Cell Technology) 1.0 ml/L After that, the pH of the medium was adjusted to 5.8. The prepared medium was dispensed in 10-15 ml each into a culture glass test tube (diameter 25 mm, height 150 mm), and then autoclaved (121, 1.2 kgf·cm ^-2 pressure, 15 minutes).

Ingredient name content Remark NH ₄ NO ₃ 1416.00 mg/L Ingredients in DKW medium MgSO ₄ 361.49 mg/L K ₂ SO ₄ 1559.00 mg/L KH ₂ PO ₄ 265.00 mg/L CaCl ₂ 112.50 mg/L Ca(NO ₃ ) ₂ 2H ₂ O 1664.64 mg/L ZnSO ₄ 7H ₂ O 17.00 mg/L Na ₂ MoO ₄ 2H ₂ O 0.39 mg/L MnSO ₄ H ₂ O 33.80 mg/L H ₃ BO ₃ 4.80 mg/L FeNaEDTA 44.63 mg/L CuSO ₄ 5H ₂ O 0.25 mg/L Glycine 2.00 mg/L Myo-Inositol 200.00 mg/L (100.00 + in DKW badge
Additional 100.00) mg/L Thiamine HCl 2.00 mg/L Ingredients contained in DKW medium Nicotinic acid 1.00 mg/L Ascorbic acid 0.1 g/L Ingredients added to DKW medium Citric acid 50 mg/L Coconut water 50 ml/L Adenine hemisulfate 25 mg/L AgNO ₃ 1 mg/L BAP 0.50 mg/L TDZ 0.10 mg/L IBA 0.10 mg/L GA ₃ 0.30 mg/L PPM 1.00 ml/L Sucrose 20.00 g/L Activated charcoal 1.00 g/L Agar 9.00 g/L PH of the medium 5.8

The internodes were placed one by one in a culture glass test tube containing the adventitious induction medium, and cultured for 2 to 3 weeks in a bright state (23±2°C, 16/8h light/dark photoperiod, 40 μmol·m ^{- 2} ). In the case of browning or blackening, in which the medium or culture turns brown or black during the culture period for 2-3 weeks, it was immediately transferred to a new medium and cultured. After culturing in the adventitious induction medium of Table 1 for 2 to 3 weeks, the rate of formation of adventitious embryos and the survival rate of internode cultures generated from the internodes were respectively investigated. Compared with one control.

As a result, as shown in Fig. 2 and Table 2, browning of cultured plants by inhibiting the action of myoinositol, adenine hemisulfate, and coconut water that promotes the growth of cultured plants in DKW medium, and phenol compounds that may occur during the culture process. Or silver nitrate, ascorbic acid, citric acid and activated carbon, which can reduce black stools, BAP and TDZ of the plant growth regulator Cytokinin, a plant growth regulator that promotes division, proliferation, growth, and differentiation of plant cells, and Auxins. It was confirmed that adventitious buds can be formed from the internodes of the jujube tree "demonstration" using a medium (Table 1) to which IBA of, and Gibberellin GA ₃ was additionally added.

Adventitious embryo formation rate (%) Culture survival rate (%) Control 5.0±0.5 ^Z 16.2±1.1 Adventitious Induction Medium 83.3±4.0 81.9±3.6

^z Each value represents the mean±SE.

< Example 2> From a negative child New Induce formation and induce development

After 2-3 weeks of initial culture, in order to germinate the adventitious embryos of Example 1 and induce the growth of shoots in earnest, the internode cultures were newly placed in the medium of Table 3 below and subcultured. The medium for growth of shoots in Table 3 is DKW medium (D0247, Duchefa) 5.6 g/L, sucrose 30 g/L, myoinositol 0.1 g/L, ascorbic acid 0.1 g/L, citric acid 0.05 g/L, coconut water 50 Ml/L, adenine hemisulfate 25 mg/L, silver nitrate 1 mg/L, activated carbon 1 g/L, agar 9 g/L, BAP 1.0 mg/L, IBA 0.2 mg/L, GA ₃ 0.3 mg/L, TDZ After mixing and preparing by adding 0.1 mg/L and 1.0 ml/L of PPM, the pH of the medium was adjusted to 5.8. Then, 50 ml each was dispensed into a plant culture vessel (250 ml in capacity, 62 mm in outer diameter, 111 mm in height), and then autoclaved (121°C, 1.2kgf· ^-2 pressure, 15 minutes). .

Ingredient name content Remark NH ₄ NO ₃ 1416.00 mg/L Ingredients contained in DKW medium MgSO ₄ 361.49 mg/L K ₂ SO ₄ 1559.00 mg/L KH ₂ PO ₄ 265.00 mg/L CaCl ₂ 112.50 mg/L Ca(NO ₃ ) ₂ 2H ₂ O 1664.64 mg/L ZnSO ₄ 7H ₂ O 17.00 mg/L Na ₂ MoO ₄ 2H ₂ O 0.39 mg/L MnSO ₄ H ₂ O 33.80 mg/L H ₃ BO ₃ 4.80 mg/L FeNaEDTA 44.63 mg/L CuSO ₄ 5H ₂ O 0.25 mg/L Glycine 2.00 mg/L Myo-Inositol 200.00 mg/L (100.00 + in DKW badge
Additional 100.00) mg/L Thiamine HCl 2.00 mg/L Ingredients contained in DKW medium Nicotinic acid 1.00 mg/L Ascorbic acid 0.1 g/L Ingredients added to DKW medium Citric acid 50 mg/L Coconut water 50 ml/L Adenine hemisulfate 25 mg/L AgNO ₃ 1 mg/L BAP 1.00 mg/L TDZ 0.10 mg/L TBA 0.20 mg/L GA ₃ 0.30 mg/L PPM 1.00 ml/L Sucrose 30.00 g/L Activated charcoal 1.00 g/L Agar 9.00 g/L pH 5.8

Internode cultures were plated one by one in a culture glass bottle containing shoot growth medium in Table 3, and cultured for 2-3 weeks in a bright state (23±2°C, 16/8h light/dark cycle, 40 μmol m ^{- 2} ). If browning or blacking occurred in the medium or culture during the culture period for 2-3 weeks, shoots were immediately transferred to a new medium and cultured. After culturing in the shoot growth medium of Table 3 for 2-3 weeks, the adventitious bud formation rate, shoot formation rate, shoot length, leaf tree, and survival rate were respectively investigated, and only the same concentration of PPM, Sucorse, and Agar were added to the basic DKW medium. It was compared with the control group cultured in.

As a result, as shown in Fig. 3 and Table 4, browning of cultured plants by inhibiting the action of myoinositol, adenine hemisulfate, and coconut water that promotes the growth of cultured plants in DKW medium, and phenol compounds that may occur during the culture process. Or silver nitrate, ascorbic acid, citric acid and activated carbon, which can reduce black stool, cytokinin-based BAP and TDZ, auxin-like IBA, and gibberellins, which are plant growth regulators that promote division, proliferation, growth, and differentiation of plant cells. Addition of GA ₃ of, but using a medium (Table 3) in which the concentrations of BAP and IBA were added twice as compared to the medium for inducing adventitious embryos (Table 1), adventitious embryos formed from the internodes of the jujube "demonstration" variety It was confirmed that germination can induce the formation of shoots.

Jeong Ah
Germination rate (%) New
Formation rate (%) New chosu
(recast) New chapter
(cm) ground game
(recast) Survival rate (%) Control 0.0 0.0 0.0 0.0 0.0 6.5 Medium for growing shoots 87.5±3.5 70.0±4.1 2.8±0.2 4.6±0.3 3.9±0.1 80.8±4.0

< Example 3> Young plant Differentiation induction

After going through the cultivation process that induces the growth of shoots formed from irregular flowers, a cultivation step is required to elongate shoots and accelerate the growth rate to differentiate into young plants (young plants).

In order to differentiate into a young plant, the shoots of Example 2 were transferred to the medium shown in Table 5 below, and cultivated for 2-3 weeks, and after 2-3 weeks, they were transferred to a new medium to induce the formation of a complete young plant. The medium for differentiation of young plants is DKW medium 5.6 g/L, sucrose 30 g/L, myoinositol 0.1 g/L, ascorbic acid 0.1 g/L, citric acid 0.05 g/L, coconut water 50 ml/L, adenine hemisulfate 25 mg/L, silver nitrate 1 mg/L, activated carbon 1 g/L, agar 9 g/L, BAP 1.0 mg/L, IBA 0.2 mg/L, GA ₃ 0.6 mg/L, TDZ 0.1 mg/L, PPM 1.0 After mixing and preparing by adding ml/L, the pH of the medium was adjusted to 5.8. Then, 80 ml each was dispensed into a plant culture vessel (500 ml capacity, 81 mm outer diameter, 132 mm height) and autoclaved (121, 1.2 kgf·cm ^-2 pressure, 15 minutes) and used. .

Ingredient name content Remark NH ₄ NO ₃ 1416.00 mg/L Ingredients contained in DKW medium MgSO ₄ 361.49 mg/L K ₂ SO ₄ 1559.00 mg/L KH ₂ PO ₄ 265.00 mg/L CaCl ₂ 112.50 mg/L Ca(NO ₃ ) ₂ 2H ₂ O 1664.64 mg/L ZnSO ₄ 7H ₂ O 17.00 mg/L Na ₂ MoO ₄ 2H ₂ O 0.39 mg/L MnSO ₄ H ₂ O 33.80 mg/L H ₃ BO ₃ 4.80 mg/L FeNaEDTA 44.63 mg/L CuSO ₄ 5H ₂ O 0.25 mg/L Glycine 2.00 mg/L Myo-Inositol 200.00 mg/L (100.00 + in DKW badge
Additional 100.00) mg/L Thiamine HCl 2.00 mg/L Ingredients contained in DKW medium Nicotinic acid 1.00 mg/L Ascorbic acid 0.1 g/L Ingredients added to DKW medium Citric acid 50 mg/L Coconut water 50 ml/L Adenine hemisulfate 25 mg/L AgNO ₃ 1 mg/L BAP 1.00 mg/L TDZ 0.10 mg/L IBA 0.20 mg/L GA ₃ 0.60 mg/L PPM 1.00 ml/L Sucrose 30.00 g/L Activated charcoal 1.00 g/L Agar 9.00 g/L pH 5.8

The shoots formed from the adventitious buds of the nodes were removed one by one in a culture glass bottle containing the culture medium for differentiation of young plants in Table 5, and each was placed in a bright state (23±2℃, 16/8h light-dark cycle, 40 μmol m ^{- 2} ) Incubated for 2-3 weeks. In the case of browning or darkening of the medium or culture medium turning brown or black during the culture period for 2-3 weeks, the shoots were immediately transferred to a new medium and cultured. The number of shoots, shoot length, leaf tree and young plant formation rate of young plants were investigated after subculture for 4-6 weeks, respectively, and the control group cultured in the medium by adding only the same concentration of PPM, Sucorse, and Agar to the basic DKW medium. Compared with.

As a result, as shown in Fig. 4 and Table 6, browning of cultured plants by inhibiting the action of myoinositol, adenine hemisulfate, and coconut water that promotes the growth of cultured plants in DKW medium, and phenol compounds that may occur during the culture process. Or silver nitrate, ascorbic acid, citric acid and activated carbon, which can reduce black stool, cytokinin-based BAP and TDZ, auxin-like IBA, and gibberellins, which are plant growth regulators that promote division, proliferation, growth, and differentiation of plant cells. Addition of GA ₃ of, but using a medium (Table 5) in which the concentration of GA ₃ was added twice as compared to the medium for inducing adventitious embryos (Table 1), stem elongation of shoots derived from adventitious embryos of the jujube tree "demonstration" It was confirmed that it can induce and differentiate into young plants.

Young plant
Formation rate (%) New chosu
(recast) New chapter
(cm) ground game
(recast) Control 0.0 0.0 0.0 0.0 Young plant
Differentiation medium 90.5±4.0 1.7±0.1 11.6±0.5 7.4±0.2

< Example 4> Plant Rooting Judo

In order to form the roots of the young plants cultured in Example 3, they were cultured using a medium prepared with the composition shown in Table 7 below. The medium for rooting young plants is DKW medium 5.6 g/L, sucrose 30 g/L, myoinositol 0.1 g/L, ascorbic acid 0.1 g/L, citric acid 0.05 g/L, coconut water 50 ml/L, adenine hemisulfate 25 mg/L, silver nitrate 1 mg/L, activated carbon 1 g/L, agar 9 g/L, BAP 0.25 mg/L, IBA 1.0 mg/L, and PPM 1.0 ml/L were added to prepare the mixture, and the pH of the medium Was adjusted to 5.8. Then, 80 ml of each was dispensed into a plant culture vessel (500 ml capacity, 81 mm outer diameter, 132 mm height) and autoclaved (121°C, 1.2kgf· ^-2 pressure, 15 minutes). .

Ingredient name content Remark NH ₄ NO ₃ 1416.00 mg/L Ingredients contained in DKW medium MgSO ₄ 361.49 mg/L K ₂ SO ₄ 1559.00 mg/L KH ₂ PO ₄ 265.00 mg/L CaCl ₂ 112.50 mg/L Ca(NO ₃ ) ₂ 2H ₂ O 1664.64 mg/L ZnSO ₄ 7H ₂ O 17.00 mg/L Na ₂ MoO ₄ 2H ₂ O 0.39 mg/L MnSO ₄ H ₂ O 33.80 mg/L H ₃ BO ₃ 4.80 mg/L FeNaEDTA 44.63 mg/L CuSO ₄ 5H ₂ O 0.25 mg/L Glycine 2.00 mg/L Myo-Inositol 200.00 mg/L (100.00 + in DKW badge
Additional 100.00) mg/L Thiamine HCl 2.00 mg/L Ingredients contained in DKW medium Nicotinic acid 1.00 mg/L Ascorbic acid 0.1 g/L Ingredients added to DKW medium Citric acid 50 mg/L Coconut water 50 ml/L Adenine hemisulfate 25 mg/L AgNO ₃ 1 mg/L BAP 0.25 mg/L IBA 1.00 mg/L PPM 1.00 ml/L Sucrose 30.00 g/L Activated charcoal 1.00 g/L Agar 9.00 g/L pH 5.8

The young plants of Example 3 were placed one by one in a culture glass bottle containing the culture medium for rooting the young plants of Table 7, and in the bright state (23±2°C, 16/8h light and dark cycle, 40 μmol m ^{- 2} ) for 4 weeks Cultured. After 4 weeks, the rooting rate, root number, root length, root diameter, and survival rate of young plants were investigated, respectively, and compared with the control group cultured in the medium by adding only the same concentration of PPM, Sucorse, and Agar to the basic DKW medium.

As a result, as shown in Fig. 5 and Table 8, browning of cultured plants by inhibiting the action of myoinositol, adenine hemisulfate, and coconut water that promotes the growth of cultured plants in DKW medium, and phenol compounds that may occur during the culture process. Alternatively, silver nitrate capable of reducing black stool, ascorbic acid, citric acid and activated carbon, cytokinin-based BAP, auxin-based IBA, which is a plant growth regulator that promotes division, proliferation, growth, and differentiation of plant cells, is additionally added, Using a medium (Table 7) in which the concentration of IBA was added five times more than the medium for differentiation of young plants (Table 5) was used, it was confirmed that root formation can be induced in the young plants of the "demonstration" variety of jujube.

Rooting rate (%) Root number
(recast) Root length (mm) Root diameter (mm) Young plant
Survival rate( % ) Control 9.0±0.3 ^Z 1.1±0.1 12.5±0.1 0.6±0.1 23.8±1.2 Young plant
Rooting badge 81.5±3.8 5.6±0.2 35.5±2.0 0.6±0.1 78.5±3.4

^z Each value represents the mean±SE.

< Example 5> Outside sublimation

Since the roots are completely formed, the young plant being cultivated in the cultivation glass bottle must 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, for purifying outside the air, the young plant of Example 4 was taken out from the culture vial, and then gently washed with water so that the culture medium does not remain on the root of the young plant, and the horticultural soil: perlite is 2:1 ) And planted in a pot (internal diameter of 110 mm, height of 113 mm) containing the mixed soil. Potted young plants are grown for 1-2 weeks in an acclimatization room similar to a culture room (23±2℃, 16/8h light/dark photoperiod, 40-50 μmol·m ^-2 , 60±5% notary humidity). , In a glass greenhouse or green house maintained at a maximum of 25℃ and a minimum of 18℃, the air humidity was gradually lowered and cured 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.

As a result, as shown in Fig. 6, it was possible to confirm the shape of a normal young seedling (drift tree) of the jujube tree'demonstration' (Fig. 6).

Claims (10)

1) inducing adventitious embryos by culturing the internodes of'Break-jo', which is a cultivar of Ziziphus jujuba Mill, in a medium for inducing adventitious embryos;
2) inducing shoot formation by culturing the adventitious embryo of step 1) in a shoot growth medium;
3) culturing the shoots of step 2) in a medium for differentiation of young plants to differentiate into young plants;
4) inducing rooting by culturing the young plant of step 3) in a medium for rooting induction; And
5) A method for forming a plant for mass proliferation of a jujube tree'demolition'comprising;
The medium for adventitious induction of step 1) is sucrose 10 to 30 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, coconut water 40 to 60 mL /L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.3 to 0.7 mg/L, TDZ 0.05 to 0.2 mg /L, IBA 0.05-0.2 mg/L, gibberellin 0.1-0.5 mg/L, and PPM 0.5-1.5 ml/L containing, plant formation method for mass propagation of jujube tree'demodulation'.
delete delete The method of claim 1, wherein the medium for growth of shoots in step 2) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, Coconut water 40-60 ml/L, adenine hemisulfate 20-30 mg/L, silver nitrate 0.5-1.5 mg/L, activated carbon 0.5-1.5 g/L, agar 7-11 g/L, BAP 0.5-1.5 mg/L , TDZ 0.05 to 0.2 mg/L, IBA 0.1 to 0.3 mg/L, Gibberellin 0.1 to 0.5 mg/L, and PPM 0.5 to 1.5 mL/L, comprising a plant formation method for mass propagation of jujube tree'demodulation'.
delete The method of claim 1, wherein the medium for differentiation of young plants in step 3) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L. , Coconut water 40 to 60 ml/L, adenine hemisulfate 20 to 30 mg/L, silver nitrate 0.5 to 1.5 mg/L, activated carbon 0.5 to 1.5 g/L, agar 7 to 11 g/L, BAP 0.5 to 1.5 mg/ L, TDZ 0.05-0.2 mg/L, IBA 0.1-0.3 mg/L, Gibberellin 0.5-0.7 mg/L, and PPM 0.5-1.5 ml/L containing, plant formation method for mass propagation of jujube tree'demodulation' .
delete The method of claim 1, wherein the medium for rooting induction of step 4) is sucrose 20 to 40 g/L, myoinositol 150 to 250 mg/L, ascorbic acid 0.05 to 0.15 g/L, citric acid 40 to 60 mg/L, Coconut water 40-60 ml/L, adenine hemisulfate 20-30 mg/L, silver nitrate 0.5-1.5 mg/L, activated carbon 0.5-1.5 g/L, agar 7-11 g/L, BAP 0.2-0.3 mg/L , IBA 0.5 to 1.5 mg / L and PPM 0.5 to 1.5 mL / L containing, plant formation method for mass propagation of jujube tree'demodulation'.
delete According to claim 1, After incubation for 1 to 2 weeks at a temperature of 20 to 25 °C and a light amount of 30 to 60 μmol m ^{- 2,} the purification outside the air in step 5) is 1 at a temperature of 15 to 28 °C. The method of forming a plant for mass proliferation of jujube trees'demodulation' that is cultured for 2 weeks.

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