KR102587569B1 - Method for culturing plant tissue of Vaccinum uliginosum L. - Google Patents

Abstract

The mass propagation and culture method of blueberry trees using sap culture of the present invention uses a medium to which a growth regulator suitable for each culture step is added, thereby lowering the survival rate of blueberry trees compared to the case of culturing in the basic medium of Anderson medium or WPM medium. We confirmed excellent results in growth characteristics such as shoot development rate, shoot length, number of shoots, rooting rate, number of roots, and root length, and planted and acclimatized plants with induced shoots, multituberculosis, and rooting in the soil according to the above method to increase survival rate and growth. It can be useful for mass propagation of this excellent blueberry tree.

Description

{Method for culturing plant tissue of Vaccinum uliginosum L.}

The present invention relates to a method for mass propagation and culture of blueberry plants using sapling culture, and specifically, by using culture of saplings where the growth point of the blueberry tree exists, to induce shoot formation of blueberry trees, induce multiple shoots, The present invention relates to a mass propagation culture method of blueberry trees that provides an optimal medium composition with excellent growth characteristics in the rooting inducing stage and exhibits excellent growth characteristics by in vitro acclimatization of seedlings.

Blueberry (Vaccinum uliginosum L.) is a deciduous small shrub belonging to the Vaccinium genus of the Ericaceae family. It reaches a height of 1 m, but in Mt. Halla it is only 10 to 15 cm. The branches are brown and young branches have or have no fine hairs. It grows naturally in alpine areas such as Yanggang-do and Hamgyeong-do in North Korea, and grows in colonies in the Samjiyeon-gun area at the foot of Baekdu Mountain, located over 1,700m above sea level. In South Korea, it is a northern cold-weather plant that rarely grows only in the highlands of Hallasan Mountain and Seorak Mountain. It has geographical significance.

The shapes of blueberry fruit vary widely, including oblate, round, and pear-shaped, and the fruit ripens to black and purple in August and September. The fruit can be collected in the fall and eaten raw, used as a raw material for juice or jam, and made into alcohol.

In foreign countries, it is used for fruit wine, juice, and pies, but due to the shortage of raw materials, its resource value is also high. In oriental medicine, it is used for a variety of medicinal purposes, including as a tonic, tonic, lowering blood sugar, strengthening capillaries, preventing worsening of night blindness and cataracts, treating scurvy, and as a thrombolytic agent. Recently, active research has been conducted on its pharmacological effects, and its antioxidant and antitumor activities. , studies on memory enhancement effects, etc. are being reported.

As a prior art regarding blueberry tree, Republic of Korea Patent No. 10-0616068 states that blueberry extract and the compounds derived from it, myricetin, rutin, hyperin, and isoquercitrin, can inhibit the activity of aldose reductase. By confirming that it exists, it is disclosed that it can be used to prevent or treat diabetes complications such as neuropathy and nephropathy. In addition, Republic of Korea Patent No. 10-0868484 confirmed the effects of blueberry fruit powder or extract powder, such as increasing skin moisture retention, reducing skin thickness and wrinkles, increasing collagen ratio, and increasing elastin amount, thereby improving skin aging caused by ultraviolet rays. It is disclosed that it can help.

As in the preceding literature, prior literature on the functional or pharmacological effects of blueberry extract or blueberry fruit has been reported, but in Korea, blueberry trees are rare because their native habitat is very limited, and no research on methods for cultivating and propagating them has been conducted. Nothing has been reported. In addition, the blueberry tree is designated as a natural monument in North Korea, and the blueberry tree on Mt. Seorak in Korea has difficulty blooming flowers but rarely bearing fruit due to climate change, so it is destined to die when the existing population disappears.

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

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

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

Accordingly, the present inventor developed a mass propagation method through tissue culture, considering that blueberry trees are difficult to propagate through seed germination or cutting methods, and developed an optimal medium composition and culture conditions suitable for mass propagation of blueberry trees in vitro. We identified plant growth regulators and developed the basic technology necessary for the cultivation of blueberry trees. We confirmed that excellent growth characteristics were exhibited during tissue culture in each stage of the medium of the present invention, and we searched for the optimal environment and soil to increase the in vitro acclimation rate. By growing and cultivating the rare plant, the blueberry tree, a mass propagation culture method was completed.

The purpose of the present invention is to provide a blueberry plant cultured by a mass propagation culture method and a mass propagation culture method of blueberry tree from sapling culture of blueberry tree, a rare plant, by shoot induction, multi-tissue induction, rooting induction and in vitro acclimation technology. do.

In order to achieve the above purpose,

This invention

1) Collecting axillary buds where the growth point of a blueberry tree exists, planting the collected buds on Anderson's medium supplemented with zeatin or 2ip to induce shoots and growing them;

2) Inducing multiple shoots by planting the grown shoots on Anderson's medium with a mixture of zeatin and IBA, Anderson's medium with a mixture of 2ip and TDZ, and Anderson's medium with a mixture of BAP and IBA. step;

3) Inducing rooting by planting the base of the multi-cultivated blueberry tree on WPM medium containing a mixture of TDZ, IBA, and activated carbon or Anderson medium containing a mixture of TDZ, IBA, and activated carbon; and

It provides a method for mass propagation and culture of blueberry trees, including the step of planting the rooting-induced blueberry plants in soil and acclimatizing them in vitro.

The mass propagation and culture method of blueberry trees using sap culture of the present invention uses a medium to which a growth regulator suitable for each culture step is added, thereby lowering the survival rate of blueberry trees compared to the case of culturing in the basic medium of Anderson medium or WPM medium. We confirmed excellent results in growth characteristics such as shoot development rate, shoot length, number of shoots, rooting rate, number of roots, and root length, and planted and acclimatized plants with induced shoots, multituberculosis, and rooting in the soil according to the above method to increase survival rate and growth. It can be useful for mass propagation of this excellent blueberry tree.

Figure 1a is a diagram showing the shoot development rate (%) during primary culture (culture for selecting basic medium) through sap culture of blueberry trees grown on WPM medium or Anderson medium.
Figure 1b is a diagram showing shoot length (mm) and number of shoots (units) during primary culture through sap culture of blueberry trees grown on WPM medium or Anderson medium.
Figure 1c is an image showing primary culture through sap culture of a blueberry planted on WPM medium or Anderson medium.
Figure 2a is a diagram showing the shoot development rate (%) during shoot induction through sap culture of blueberry trees grown on Anderson's medium supplemented with zeatin or 2ip.
Figure 2b is a diagram showing shoot length (mm) and number of shoots (units) during shoot induction through sap culture of blueberry trees grown on Anderson's medium supplemented with zeatin or 2ip.
Figure 2c is an image showing shoot induction through sap culture of a blueberry tree grown on Anderson's medium supplemented with zeatin or 2ip.
Figure 3a shows the multiple shoot occurrence rate (%) through multiple shoot induction of blueberry shoots grown on Anderson's medium mixed with zeatin and IBA, Anderson's medium with mixed 2ip and TDZ, and Anderson's medium mixed with BAP and IBA. This is the diagram shown.
Figure 3b shows shoot length (mm) through multiple shoot induction of blueberry shoots grown on Anderson's medium mixed with zeatin and IBA, Anderson's medium with mixed 2ip and TDZ, and Anderson's medium mixed with BAP and IBA. This is a diagram showing the number of shoots.
Figure 3c is an image showing multi-stage induction of blueberry shoots grown on Anderson's medium containing a mixture of zeatin and IBA, Anderson's medium containing a mixture of 2ip and TDZ, and Anderson's medium containing a mixture of BAP and IBA.
Figure 4a is a diagram showing the rooting rate (%) through rooting induction of the base of blueberry multi-tissues grown on WPM medium mixed with TDZ, IBA, and activated carbon or Anderson medium mixed with TDZ, IBA, and activated carbon.
Figure 4b shows the root length (mm) and root number (mm) through rooting induction of the base of a blueberry multi-tissue grown on WPM medium mixed with TDZ, IBA, and activated carbon or Anderson medium mixed with TDZ, IBA, and activated carbon ( This is a diagram showing a dog).
Figure 4c is an image showing the rooting of the base of a blueberry multi-tissue grown on WPM medium containing a mixture of TDZ, IBA, and activated carbon or Anderson's medium containing a mixture of TDZ, IBA, and activated carbon.
Figure 5a is a diagram showing the survival rate (%) by soil after acclimatization of blueberry trees.
Figure 5b is a diagram showing the number of roots (number) for each soil after purification of blueberry trees.
Figure 5c is a diagram showing the root length (mm) for each soil after purification of blueberry trees.
Figure 5d is an image showing the appearance of acclimatized seedlings in each soil 4 weeks after acclimatization of blueberry trees.

Hereinafter, the present invention will be described in detail.

This invention

1) Collecting axillary buds where the growth point of a blueberry tree exists, planting the collected buds on Anderson's medium supplemented with zeatin or 2ip to induce shoots and growing them;

2) Inducing multiple shoots by planting the grown shoots on Anderson's medium with a mixture of zeatin and IBA, Anderson's medium with a mixture of 2ip and TDZ, and Anderson's medium with a mixture of BAP and IBA. step;

3) Inducing rooting by planting the base of the multi-tissue induced blueberry tree on WPM medium containing a mixture of TDZ, IBA, and activated carbon or Anderson medium containing a mixture of TDZ, IBA, and activated carbon; and

4) planting the rooting-induced blueberry plants in soil and acclimatizing them in vitro; providing a culture method for mass propagation of blueberry trees, comprising:

The axillary bud may be selected and collected from the group consisting of branches or stems, but preferably, branches of the plant may be collected and used.

The growing point is the part that creates new stems and leaves, and is usually located in the normal bud or axillary bud of the plant, and is also present at the tip of the root. In other words, the growing point is the youngest part of the shoot, which has not yet differentiated into stems and leaves, and is where the apical meristem of the shoot is located.

The growing point is usually shaped like a mountain called a 'dome', and leaves develop next to it. However, the shape of the growing point is very diverse, ranging from a high dome shape to the usual low dome shape, and also from a flat shape to a somewhat concave shape. Therefore, the height and width of the growing point are also very diverse. Although the standards for height and width are not clearly defined, the height of the growing point generally refers to the height of the upper part where the youngest leaf is attached, and the area refers to the diameter at the attached part.

The method of cultivating a blueberry plant according to the present invention may include the step of inducing shoots by pricking the sap portion where the growth point of the blueberry tree is located on a culture medium.

As used herein, the term "culture medium" refers to a medium used in the step of inducing shoots from the sap portion of a blueberry tree, and includes general basic media that can be used to induce shoots from plant tissue for tissue culture. . The culture medium can be artificially prepared and used, or a commercially available one can be used.

Specific examples of the commercially available culture media include MS (Murashige and Skoog basal), WPM (woody plant medium), B5 (Gamborg B5), W (White), LMWP (Lloyd and McCown Woody Plant basal), VW (Vacin and Went), Km (Knudson C modified orchid), M (Mitra replate/maintenance), NN (Nitsch and Nitsch), Anderson, BDS (modified B5 of Dunstan and Short), and SH (Schenk & Hildebrandt basal salt) media. , preferably WPM medium or Anderson medium can be used.

The medium used in the present invention can be based on Anderson's medium.

The Anderson medium is a medium developed to cultivate shoots of plants of the genus Rhododendron and is used as a basic medium during plant tissue culture.

Additionally, the medium used in the present invention can be based on WPM (woody plant mediem) medium.

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

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

When preparing and using the culture medium, the culture medium may contain at least one selected from the group consisting of carbon sources, macro elements, trace elements, vitamins, and amino acids. The carbon source may be any one or more selected from the group consisting of sucrose, glucose, maltose, lactose, and fructose, and may specifically be sucrose. The carbon source may be included at a concentration of 20 to 40 g/L, specifically 23 to 37 g/L, and more specifically 26 to 33 g/L. The bulk elements include MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KH ₂ PO ₄ , Ca(NO) ₂ ·4H ₂ O, K ₂ SO ₄ , KCl, KI, KNO ₃ and At least one selected from the group consisting of NaH ₂ PO ₄ ·H ₂ O, specifically Anderson's medium is MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KI, KNO ₃ and At least one selected from the group consisting of NaH ₂ PO ₄ ·H ₂ O, more specifically MgSO ₄ ·7H ₂ O, CaCl ₂ ·2H ₂ O, NH ₄ NO ₃ , KI, KNO ₃ and NaH ₂ PO ₄ · It may contain H ₂ O.

In the Anderson medium, MgSO ₄ ·7H ₂ O may be included at a concentration of 340 to 400 mg/L, specifically 350 to 390 mg/L, and more specifically 360 to 380 mg/L. The CaCl ₂ ·2H ₂ O may be included at a concentration of 410 to 470 mg/L, specifically 420 to 460 mg/L, and more specifically 430 to 450 mg/L. The NH ₄ NO ₃ may be included at a concentration of 370 to 430 mg/L, specifically 380 to 420 mg/L, and more specifically 390 to 410 mg/L. Additionally, the KI may be included at a concentration of 0.05 mg/L to 0.55 mg/L, specifically 0.1 mg/L to 0.5 mg/L, and more specifically 0.2 mg/L to 0.4 mg/L. Additionally, the KNO ₃ may be included at a concentration of 450 to 510 mg/L, specifically 460 to 500 mg/L, and more specifically 470 to 490 mg/L. The NaH ₂ PO ₄ ·H ₂ O may be included at a concentration of 140 to 200 mg/L, specifically 150 to 190 mg/L, and more specifically 160 to 180 mg/L.

Additionally, in the WPM medium, MgSO ₄ ·7H ₂ O may be included at a concentration of 340 to 400 mg/L, specifically 350 to 390 mg/L, and more specifically 360 to 380 mg/L. The CaCl ₂ ·2H ₂ O may be included at a concentration of 76 to 116 mg/L, specifically 86 to 106 mg/L, and more specifically 90 to 102 mg/L. The NH ₄ NO ₃ may be included at a concentration of 370 to 430 mg/L, specifically 380 to 420 mg/L, and more specifically 390 to 410 mg/L. KH ₂ PO ₄ may be included at a concentration of 140 to 200 mg/L, specifically 150 to 190 mg/L, and more specifically 160 to 180 mg/L. Ca(NO) ₂ ·4H ₂ O may be included at a concentration of 530 to 580 mg/L, specifically 540 to 570 mg/L, and more specifically 550 to 560 mg/L. Additionally, K ₂ SO ₄ may be included at a concentration of 960 to 1,020 mg/L, specifically 970 to 1,010 mg/L, and more specifically 980 to 1,000 mg/L.

The trace elements are FeSO ₄ ·7H ₂ O, MnSO ₄ ·H ₂ O, ZnSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, H ₃ BO ₃ , Na ₂ -EDTA, Na ₂ MoO ₄ ·2H ₂ O and CoCl ₂ ·6H ₂ O, specifically Anderson's medium is FeSO ₄ ·7H ₂ O, ZnSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, H ₃ BO ₃ , Na ₂ MoO ₄ ·2H ₂ O and CoCl ₂ ·6H ₂ O, more specifically FeSO ₄ ·7H ₂ O, ZnSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, It may include H ₃ BO ₃ , Na ₂ MoO ₄ ·2H ₂ O and CoCl ₂ ·6H ₂ O.

In the Anderson medium, FeSO ₄ ·7H ₂ O may be included at a concentration of 30 to 80 mg/L, specifically 40 to 70 mg/L, and more specifically 50 to 60 mg/L, and the ZnSO ₄ ·7H ₂ O May be included at a concentration of 6 to 11 mg/L, specifically 7 to 10 mg/L, and more specifically 8 to 9 mg/L. The CuSO ₄ ·5H ₂ O may be contained at a concentration of 0.005 to 0.045 mg/L, specifically 0.015 to 0.035 mg/L, and more specifically 0.02 to 0.03 mg/L, and the H ₃ BO ₃ may be contained at a concentration of 4 to 9 mg/L. /L, specifically 5 to 8 mg/L, more specifically 6 to 7 mg/L. In addition, the Na ₂ MoO ₄ ·2H ₂ O may be included at a concentration of 0.05 to 0.45 mg/L, specifically 0.15 to 0.35 mg/L, and more specifically 0.2 to 0.3 mg/L, and CoCl ₂ ·6H ₂ O It may be included at a concentration of 0.005 to 0.0045 mg/l, specifically 0.0015 to 0.0035 mg/l, and more specifically 0.002 to 0.003 mg/l.

In the WPM medium, FeSO ₄ ·7H ₂ O may be included at a concentration of 10 to 40 mg/L, specifically 15 to 35 mg/L, and more specifically 20 to 30 mg/L, and the MnSO ₄ ·H ₂ O It may be included at a concentration of 10 to 35 mg/L, specifically 15 to 30 mg/L, and more specifically 20 to 25 mg/L. The ZnSO ₄ ·7H ₂ O may be included at a concentration of 6 to 11 mg/L, specifically 7 to 10 mg/L, and more specifically 8 to 9 mg/L, and the CuSO ₄ ·5H ₂ O may be contained at a concentration of 0.05 to 10 mg/L. It may be included at a concentration of 0.45 mg/L, specifically 0.15 to 0.35 mg/L, and more specifically 0.2 to 0.3 mg/L. The H ₃ BO ₃ may be included at a concentration of 4 to 9 mg/L, specifically 5 to 8 mg/L, and more specifically 6 to 7 mg/L. In addition, the Na ₂ -EDTA may be included at a concentration of 25 to 50 mg/L, specifically 30 to 44 mg/L, and more specifically 34 to 40 mg/L, and the Na ₂ MoO ₄ ·2H ₂ O It may be included at a concentration of 0.05 to 0.45 mg/L, specifically 0.15 to 0.35 mg/L, and more specifically 0.2 to 0.3 mg/L.

The vitamins and amino acids may include one or more selected from the group consisting of glycine, inositol, nicotinic acid, pyridoxine, and thiamine, specifically glycine, inositol, nicotinic acid, pyridoxine, and thiamine.

In the Anderson medium, thiamine may be included at a concentration of 0.1 to 0.7 mg/L, specifically 0.2 to 0.6 mg/L, and more specifically 0.3 to 0.5 mg/L.

In the WPM medium, glycine may be contained at a concentration of 0.5 to 3.5 mg/L, specifically 1.0 to 3.0 mg/L, more specifically 1.5 to 2.5 mg/L, and the inostol may be contained at a concentration of 70 to 130 mg/L, specifically It may be included at a concentration of 80 to 120 mg/L, more specifically 90 to 110 mg/L. The nicotinic acid may be contained at a concentration of 0.2 to 0.8 mg/L, specifically 0.3 to 0.7 mg/L, and more specifically 0.4 to 0.6 mg/L, and the pyridoxine may be contained at a concentration of 0.2 to 0.8 mg/L, specifically 0.3 to 0.7 mg/L. It may be included at a concentration of mg/L, more specifically 0.4 to 0.6 mg/L. Additionally, the thiamine may be included at a concentration of 0.4 to 1.6 mg/L, specifically 0.6 to 1.4 mg/L, and more specifically 0.8 to 1.2 mg/L.

The mixed medium may be adjusted to pH 3.5 to 6.0, specifically pH 4.0 to 5.8, and more specifically pH 4.5 to 5.5.

The culture medium may be prepared as a liquid medium or a solid medium depending on the purpose of the culture, and when prepared as a solid medium, components such as agar or gelite may be additionally added.

The Andersen medium in step 1) can be prepared by single addition of 0.1 to 5.0 mg/L of zeatin, preferably by single addition of 0.5 to 3.0 mg/L of zeatin, and more preferably It can be prepared by single addition of 0.5 to 1.5 mg/L of atin.

The Andersen medium in step 1) can be prepared by single addition of 2ip 1.0 to 12 mg/L, preferably 2ip 3.0 to 10 mg/L, and more preferably 2ip 4.0 to 6.0 mg. It can be prepared by simply adding /L.

Anderson's medium containing a mixture of zeatin and IBA in step 2) can be prepared by adding a mixture of 0.1 to 2.0 mg/L of zeatin and 0.05 to 1.0 mg/L of IBA, but preferably 0.3 mg/L of zeatin. It can be prepared by mixing 0.1 to 1.8 mg/L of zeatin and 0.1 to 0.8 mg/L of IBA, and more preferably, mixing 0.5 to 1.5 mg/L of zeatin and 0.3 to 0.7 mg/L of IBA. .

Anderson's medium containing a mixture of 2ip and TDZ in step 2) can be prepared by adding a mixture of 2ip 3.0 to 7.0 mg/L and TDZ 0.01 to 3.0 mg/L, but 2ip 3.5 to 6.5 mg/L and TDZ 0.03 to 3.0 mg/L. It can be manufactured by mixing 2.0 mg/L, and more preferably by mixing 4.0 to 6.0 mg/L of 2ip and 0.05 to 0.15 mg/L of TDZ.

Anderson's medium containing a mixture of BAP and IBA in step 2) can be prepared by adding a mixture of 0.1 to 2.0 mg/L of BAP and 0.01 to 1.0 mg/L of IBA, but preferably 0.5 to 1.5 mg/L of BAP and It can be manufactured by mixing 0.1 to 0.5 mg/L of IBA.

The WPM medium containing a mixture of TDZ, IBA, and activated carbon in step 3) can be prepared by adding a mixture of 0.01 to 1.00 mg/L of TDZ, 0.5 to 2.5 mg/L of IBA, and 0.1 to 2.0 g/L of activated carbon, but is preferred. It can be prepared by adding a mixture of 0.05 to 0.1 mg/L of TDZ, 0.7 to 2.0 mg/L of IBA, and 0.8 to 1.8 g/L of activated carbon, more preferably 0.06 to 0.08 mg/L of TDZ and 1.0 to 2.0 of IBA. It can be manufactured by adding a mixture of mg/L and activated carbon of 0.5 to 1.5 g/L.

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

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

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

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

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

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

In addition, the present invention is 100% non-fertilized topsoil, non-fertilized topsoil: peat moss (5:5), non-fertilized topsoil: peat moss (7:3), non-fertilized topsoil: peat moss (3:7), 100% fertilized topsoil, fertilized topsoil when acclimatized in vitro. : Peat moss (5:5), peat moss: peat moss (7:3), peat moss (7:3), peat moss (3:7), soil with 100% peat moss can be used, but most preferably, soil with 100% peat moss is used. .

Peat moss is currently the most widely used organic material in the world. About 98% of its volume is made up of water cells, so it contains water and air in an ideal ratio, providing excellent breathability and water retention. In addition, because it has a COOH ^- group on the cell surface, it has a large cation exchange capacity and therefore has good water retention capacity. It is in the state of organic carbon that is stable against decomposition, so decomposition occurs slowly in the medium, so the physicochemical properties can be maintained for a long time. In addition, the content of inorganic components is very low and it is difficult to decompose, so there is not much elution of inorganic components during the decomposition process, so it is easy to control fertilization. It is produced in a humid place in cold regions, so it is free from mold, bacteria, pests and weed seeds, and is light to handle. Not only is it easy to use, but because it is fibrous, it has excellent self-bonding strength and has good formability (Kim I-yeol, 2003), and due to low soil acidity, it is an easy material for the acclimatization and cultivation of plants that grow well in low acidity, such as blueberry trees.

Blueberry seedlings cultured and grown in each medium of the present invention exhibit excellent growth characteristics, such as a survival rate of 90 to 100%, a shoot length of 60 to 75 mm, a leaf length of 7 to 10 mm, and a leaf width of 5 to 6 mm when acclimatized in vitro in 100% peat moss.

In addition, the present invention can provide blueberry trees grown by the blueberry mass propagation culture method of the present invention.

In a specific example of the present invention, the present inventor used Anderson's medium and WPM medium as base media to perform in vitro culture proliferation tests of blueberry (Table 1). As a result of seedlings collected from blueberry branches and initial culture on Anderson medium and WPM medium, the results showed better shoot development rate, shoot length, and number of shoots. However, it is recommended to use Anderson medium when culturing blueberry trees in vitro. Suitability was confirmed (Table 2, Figures 1a, 1b, and 1c).

As a result of checking the shoot development rate, shoot length, and number of shoots on Anderson's medium to which the growth regulator zeatin or 2ip was added singly to induce shoots, the growth characteristics of the treatment group in which 1.0 mg/L of zeatin was added to Anderson's medium were found to be better. was the best, and the treatment in which 5.0 mg/L of 2ip was added to Anderson's medium was confirmed to have the next best growth characteristics (Table 3, Figures 2a, 2b, and 2c).

As a medium for inducing multiple shoots in the shoots of the blueberry tree, Anderson's medium containing a mixture of 2ip and TDZ treated with different concentrations of growth regulators, Anderson's medium containing a mixture of BAP and IBA, and zeatin and IBA were used. As a result of using a mixed Andersen medium, the Andersen medium mixed with 1.0 mg/L of zeatin and 0.5 mg/L of IBA showed the best growth characteristics with 100% shoot development rate, 10.7±5.3 mm shoot length, and 5.3±0.5 shoot numbers. (Table 4, Figures 3a, 3b and 3c).

Among the media prepared to induce rooting of the base of the multi-tuberculosis-induced blueberry tree, the rooting rate was 100%, and the rooting rate was 100% in the treatment group in which 1.0 g/L of activated carbon, 0.07 mg/L of TDZ, and 2.0 mg/L of IBA were added mixed to the WPM medium. It showed the best root development results with a number of 13.7 ± 6.7 roots and a root length of 9.3 ± 3.1 mm (Table 5, Figures 4a, 4b and 4c).

When the in-plant cultured blueberry plants were acclimatized in vitro in 100% peat moss, it was confirmed that the growth characteristics were the best among all treatments, with a survival rate of 100%, shoot length of 67.1 mm, leaf length of 8.7 mm, and leaf width of 5.5 mm (Table 6, Figure 5a, 5b, 5c and 5d)

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

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

<Example 1> Collection, cleaning and disinfection of blueberry sap

In a specific example of the present invention, the present inventor collected and used axial buds (lateral buds that differentiate into leaves in the same year) where the growing point of the blueberry tree exists. The collected fluid was washed under running tap water for 5 minutes, sterilized with 70% ethanol for 3 minutes, and washed three times with distilled water. The sterilized liquid was sterilized for 30 minutes by adding 2-3 drops of Tween 20 to 2.5% sodium hypochlorite (NaCIO) solution in a clean bench, and then washed 5 times with sterilized water.

<Example 2> Preparation of basic medium for in vitro culture of blueberry

The medium used in the in vitro culture growth test of blueberry was Anderson (1984) and WPM (Lloyd and McCown, 1981) powder medium as the basic medium (Table 1). After adjusting the pH of the medium to 5.0, 8 ml of the medium prepared with 8% agar was dispensed into a test tube and sterilized in a high-pressure sterilizer at 121°C for 15 minutes. The culture room conditions were 16 hours of daylight and 8 hours of darkness under 2,000 lux of light, and the culture temperature was 23 ± 1°C.

Composition (mg/L) anderson badge WPM badge macroelement MgSO₄·7H ₂ O 370 370 CaCl₂·2H ₂ O 440 96 NH₄NO ₃ 400 400 KH ₂ PO ₄ - 170 Ca(NO)₂·4H ₂ O - 556 K₂SO₄ 990 KCl - - KI 0.3 - KNO₃ 480 - NaH₂PO₄·H₂O 170 - Trace elements FeSO₄·7H₂O 55.7 27.8 MnSO₄·H₂O - 22.3 ZnSO₄·7H₂O 8.6 8.6 CuSO₄·5H₂O 0.025 0.25 H₃BO₃ 6.2 6.2 Na₂-EDTA - 37.3 Na₂MoO₄·2H₂O 0.25 0.25 CoCl₂·6H₂O 0.0025 - vitamins and amino acids Glycine - 2.0 Inositol - 100 Nicotinic acid - 0.5 Pyridoxine·HCl - 0.5 Thiamine·HCl 0.4 1.0 Sucrose 30,000 30,000 Agar 8,000 8,000 pH 5.0 5.0

<Example 3> Initial culture of blueberry saplings

Three saplings collected from blueberry branches were planted three times each on Anderson's medium and WPM medium without the addition of growth regulators, and the shoot development rate, shoot length, and number of shoots were examined after 6 weeks (Table 2, Figure 1a, Figure 1b and Figure 1c). After 3 weeks of rooting, shoots occurred on both Anderson and WPM media. In the WPM medium, when 9 seedlings were formed, the shoot development rate was 11.1% and only 1 shoot was formed, whereas in the Anderson medium, 9 seedlings were produced. When wounded, the shoot development rate was 33.3% and 3 shoots were generated (Table 2 and Figure 1a). As a result of the examination 6 weeks after the tooth was formed, the growth of new shoots in Anderson medium was 7.1 ± 4.6 mm and the number of shoots was 1.0 ± 0.0, while in the WPM medium, the shoot length was 2.5 ± 0.0 mm and the number of shoots was 1.0 ± 0.0. It was shown that it is suitable to use Anderson's medium for in-flight culture of blueberry trees (Table 2 and Figure 1b), and the growth of blueberry saplings was confirmed by primary culture on Anderson's medium and WPM medium. There is (Figure 1c).

badge Chi Sang number of shoots
(dog) incidence rate
(%) Shoot length
(cm) number of shoots
(dog) Anderson 9 3 33.3% 7.1±4.6 1.0±0.0 W.P.M. 9 One 11.1% 2.5±0.0 1.0±0.0

<Example 4> Shoot induction through sap culture of Blueberry tree

In order to identify a growth regulator for efficient shoot induction, the present inventors used zeatin [6-(4-hydroxy-3-methyl-2-butenylamino)purine], a cytokinin-based growth regulator, on Anderson's medium. Add single-use at different concentrations (1.0, 1.5, 2.0, 3.0 mg/L), and add single-use 2ip[(N6-(2-isopentyl)adenine)] at different concentrations (3, 5, 8, 10 mg/L). Blueberry saplings were planted in one treatment group and observed for 6 weeks, and the shoot occurrence rate and growth status (shoot length and number of shoots) according to the treatment group were investigated (Table 3, Figures 2a, 2b, and 2c). First, 8 days after planting the blueberry saplings on the medium, the development of shoots was first observed in the treatment group in which 1.0 mg/L of zeatin was added to Anderson's medium, and in the second week after planting, the control group (without the addition of growth regulator) The development of shoots was observed in all treatments except Anderson's medium). As a result of examination 6 weeks after tooth formation, no shoots occurred in Andersen's medium (control) without the addition of growth regulator, but in the treatment group with 1.0 mg/L of zeatin added to Anderson's medium, shoot development rate was 100%, shoot length was 13.7 ± 6.7 mm, The best results were obtained with a number of shoots of 1.1 ± 0.3 (Table 3, Figures 2a and 2b). Next, the treatment group in which 5.0 mg/L of 2ip was added to Anderson's medium showed good results with 100% shoot development rate, shoot length of 5.1 ± 2.8 mm, and shoot number of 1.0 ± 0.0 (Table 3, Figures 2a and 2b). . Next, in the treatment group in which 2.0 mg/L of zeatin was added to Anderson's medium, the shoot development rate was 100%, the shoot length was 3.3 ± 1.7 mm, and the number of shoots was 1.0 ± 0 (Table 3, Figures 2a and 2b). ). The treatment that showed the lowest results was the treatment in which 10.0 mg/L of 2iP was added to Anderson's medium, with a shoot development rate of 55.6%, shoot length of 3.5 ± 2.4 mm, and number of shoots of 1.0 ± 0 (Table 3, Figures 2a and 2a). Figure 2b).

Based on the results of this experiment as described above, the most suitable conditions for shoot induction of blueberry trees are when 1.0 mg/L of zeatin is added to Anderson's medium, and the shoot length becomes shorter as the concentration of zeatin is increased. A trend was observed (Table 3 and Figure 2b). 2iP showed the highest shoot development rate (%) when 5.0 mg/L was added, and when added at a higher concentration, the shoot development rate tended to decrease (Table 3, Figures 2a and 2b), and Anderson ( As a result of adding different concentrations of zeatin or 2ip to Anderson) medium, it was confirmed that the shoot length was longest when 1.0 mg/L of zeatin was added (Figure 2c).

growth regulator Chi Sang Number of shoots
(dog) incidence rate
(%) Shoot length
(mm) number of shoots
(dog) Anderson (0mg/L, control) 9 0 0.0 0.0±0.0 0.0±0.0 Zeatin (1.0mg/L) 9 9 100 13.7±6.7 1.1±0.3 Zeatin (1.5mg/L) 9 8 88.9 8.6±6.2 1.0±0.0 Zeatin (2.0mg/L) 9 9 100 3.3±1.7 1.0±0.0 Zeatin (3.0mg/L) 9 7 77.8 2.7±1.5 1.0±0.0 2ip(3.0mg/L) 9 5 55.6 7.0±5.2 1.0±0.0 2ip(5.0mg/L) 9 9 100 5.1±2.8 1.0±0.0 2ip(8.0mg/L) 9 6 66.7 5.8±5.7 1.0±0.0 2ip(10.0mg/L) 9 5 55.6 3.5±2.4 1.0±0.0

<Example 5> Induction of multi-cultivation of blueberry tree

To induce multiple shoots in induced blueberry shoots, 5.0 mg/L of cytokinin growth regulator 2ip ((N6-(2-isopentyl)adenyl) and TDZ (1-phenyl-3) were added to Anderson's medium. -(1,2,3-thiadiazol-5-yl)urea) mixed at each concentration (0.05, 0.1, 0.15 mg/L), BAP (N6-benzyl-adenine) 1.0 mg/L and IBA (4-(3-indolyl)butyric acid) mixed and added at different concentrations (0.1, 0.3, 0.5mg/L), zeatin [6-(4-hydroxy-3-methyl-2-butenylamino) [Purine] 1.0 mg/L and IBA (0.1, 0.3, 0.5 mg/L) were added in a mixture of each concentration, and 3 shoots were germinated 3 times each and observed for 6 weeks, and the shoot occurrence rate and growth status according to the treatment group were observed. The results of examining (shoot length and shoot number) are as follows (Table 4, Figures 3a, 3b and 3c).

Eight days after planting the blueberry shoots on the medium, 1.0 mg/L of zeatin and 0.3 mg/L of IBA were added to Anderson's medium, and 1.0 mg/L of zeatin and 0.5 mg/L of IBA were added to the treatment group. The development of shoots was observed in one treatment group, and the growth survey results after 6 weeks of planting showed 100% shoot development rate and 100% shoot length in Anderson medium mixed with 1.0 mg/L of zeatin and 0.5 mg/L of IBA. The best results were obtained with a diameter of 10.7 ± 5.3 mm and a number of shoots of 5.3 ± 0.5 (Table 4, Figures 3a and 3b). The next treatment that showed good results was the treatment in which 2ip 5.0mg/L and TDZ 0.1mg/L were mixed, showing good results with 100% shoot development rate, shoot length of 3.5±1.8mm, and number of shoots of 2.0±0.9.

Among the treatments excluding the control, the treatment that showed the lowest results was the treatment in which 1.0 mg/L of BAP and 0.5 mg/L of IBA were mixed, with a shoot occurrence rate of 11%, shoot length of 4.9 ± 1.7 mm, and number of shoots of 4.0 ± 0.0. Although the development was good, the shoot development rate was found to be very low (Table 4, Figures 3a and 3b).

In this experiment, the most suitable conditions for multiple induction of blueberry trees were when 1.0 mg/L of zeatin + 0.5 mg/L of IBA were added to Anderson's medium, and the trend according to the addition of growth regulators was as follows. When 1.0 mg/L of zeatin and IBA were mixed at different concentrations (0.1, 0.3, 0.5 mg/L), the higher the concentration of IBA, the better the shoot development rate and multiple shoot induction status (shoot number, shoot length). (Table 4, Figures 3a and 3b), and it can be seen that the number of shoots was the highest in the treatment group in which 1.0 mg/L of zeatin + 0.5 mg/L of IBA was mixed (Figure 3c).

growth regulator Chi Sang Number of shoots
(dog) incidence rate
(%) Shoot length
(mm) number of shoots
(dog) Anderson (0mg/L, control) 9 2 22.2 3.3±0.5 2.0±0.0 2ip+TDZ(5.0mg/L)(0.05mg/L) 9 7 77.8 4.0±2.8 3.0±0.8 2ip+TDZ(5.0mg/L)(0.1mg/L) 9 97 100 3.5±1.8 2.0±0.9 2ip+TDZ(5.0mg/L)(0.15mg/L) 9 9 100 3.2±1.8 1.9±0.6 BAP+IBA(1.0mg/L)(0.1mg/L) 9 3 33.3 6.8±3.4 2.0±1.0 BAP+IBA(1.0mg/L)(0.3mg/L) 9 3 33.3 4.5±1.3 1.3±0.6 BAP+IBA(1.0mg/L)(0.5mg/L) 9 One 11.1 4.9±1.7 4.0±0.0 Zeatin + IBA (1.0mg/L) (0.1mg/L) 9 5 55.6 6.8±5.0 1.6±0.9 Zeatin + IBA (1.0mg/L) (0.3mg/L) 9 5 55.6 8.9±6.4 2.0±1.0 Zeatin + IBA (1.0mg/L) (0.5mg/L) 9 9 100 10.7±5.3 5.3±0.5

<Example 6> Induction of rooting of blueberry multi-tissue base

To induce rooting, shoots cut from the base of the multi-tissue induced blueberry were raised. As a control group, Anderson medium and WPM medium without growth regulators were used as basic media, and as a test group, basic media was used. A total of 1.0 g/L of activated carbon was added. As a growth regulator added to the test group, 0.07 mg/L of cytokinin-based TDZ and auxin-based IBA were mixed at different concentrations (1.0, 1.5, 2.0 mg/L) to create a medium, and then placed in three test tubes. The wound was repeated three times. After observing for 8 weeks, the rooting rate and growth status (root length, number of roots, etc.) according to treatment were examined and the results are as follows (Table 5, Figures 4a, 4b and 4c).

First, the development of roots could be observed in all treatments except for WPM (0mg/L, control) medium and Anderson (0mg/L, control) medium without addition of growth regulators from 3 weeks after the rooting period. As a result of the investigation after 8 weeks, the treatment that showed the best root development was the treatment in which 0.07 mg/L of TDZ and 2.0 mg/L of IBA were added to the WPM medium, with 100% rooting rate, 13.7 ± 6.7 roots, and 9.3 ± 3.1 root length. It showed the best root development results in mm, followed by a treatment in which 0.07 mg/L of TDZ and 1.5 mg/L of IBA were added to the WPM medium, with a rooting rate of 100%, number of roots 12.3±7.7, and root length 9.2± Excellent results were shown at 3.0 mm (Table 5, Figures 4a and 4b). Among the treatments in which growth regulators were added to the basic medium, the treatment that showed the poorest results was the treatment in which 0.07 mg/L of TDZ and 1.0 mg/L of IBA were added to Anderson's medium, with a rooting rate of 55.6% and the number of roots: 2.4 ± 1.5. , the root length was found to be 3.1 ± 1.3 mm (Table 5, Figures 4a and 4b). During primary culture, shoot induction, and multiple shoot induction, the Anderson medium had better shoot growth than the WPM medium, but during rooting induction, the growth regulator was added to the Anderson medium and WPM medium at the same concentration. When using WPM medium, better results were obtained. It can be seen that when WPM medium was treated with different concentrations of the growth regulator, more roots were developed than when cultured on Anderson medium with the growth regulator (FIG. 4c).

growth regulator Chi Sang number of roots
(dog) Rooting rate
(%) root length
(mm) number of roots
(dog) W.P.M.
(0mg/L, control group) 9 3 33.3 2.0±0.3 1.3±1.8 WPM+TDZ (0.07mg/L)+IBA (1.0mg/L)+Activated Carbon (1.0g/L) 9 8 88.9 8.0±3.3 9.1±6.1 WPM+TDZ (0.07mg/L)+IBA (1.5mg/L)+Activated Carbon (1.0g/L) 9 9 100 9.2±3.0 12.3±4.7 WPM+TDZ (0.07mg/L)+IBA (2.0mg/L)+Activated Carbon (1.0g/L) 9 9 100 9.3±3.1 13.7±6.7 Anderson 9 0 0 - - Anderson+TDZ(0.07mg/L)+IBA
(1.0mg/L)+Activated carbon (1.0g/L) 9 5 55.6 3.1±1.3 2.4±1.5 Anderson+TDZ (0.07mg/L)+IBA (1.5mg/L)+Activated Carbon (1.0g/L) 9 7 77.8 3.9±1.5 2.4±1.6 Anderson+TDZ (0.07mg/L)+IBA (2.0mg/L)+Activated Carbon (1.0g/L) 9 7 77.8 3.6±1.3 2.7±1.5

<Example 7> In vitro acclimation of blueberry plants

The process of physiologically adapting a plant cultured in-flight to a new environment or climatic condition is called acclimation. It is carried out naturally or artificially and is subject to complex environmental factors such as seasonal changes and specific environments such as light intensity. Like factors, it occurs within a limited range to which plants can adapt (Baek Ki-yeop, 2001)

In order to adapt to the external environment and determine appropriate soil conditions for forage transplantation of blueberry trees that had been induced to root through tissue culture before soil transplantation, plants rooted in the air were placed in acclimatization boxes (25cm × 12cm × 23cm). 100% non-fertilized topsoil, non-fertilized topsoil: peat moss (5:5), non-fertilized topsoil: peat moss (7:3), non-fertilized topsoil: peat moss (3:7), 100% fertilized topsoil, fertilized topsoil: peat moss (5:5), They were planted by filling them with 100% peat moss or soil mixed at a weight ratio of fertilized topsoil: peat moss (7:3) and fertilized topsoil: peat moss (3:7). The environment of the acclimatization room was acclimatized under artificial light conditions with an illumination intensity of 2,000 lux, and the day length was set to 10 hours of light and 14 hours of darkness. The temperature in the laboratory was maintained at a constant temperature of 25°C by operating a thermohygrostat, and the humidity was maintained by covering it with a 5 mm transparent acrylic plate. After a week, the acrylic plate was opened to allow air to pass through, and the temperature was maintained 2 to 3 times a week. The soil was watered with a spray to prevent it from drying out, and the survival rate and growth status (shoot length, number of shoots, root length, number of roots, etc.) were examined 4 weeks after the acclimatization test (Table 6, Figures 5a, 5b, 5c and 5d). .

As a result of the acclimatization treatment after 4 weeks, the treatment that showed the best results was the treatment that was acclimated to 100% peat moss, with a survival rate of 100%, shoot length of 67.1mm, leaf length of 8.7mm, and leaf width of 5.5mm, which was the best among all treatments, and the number of roots was 7.2. 50% fertilizer-free topsoil: It was 0.2 smaller than 50% peat moss, but there was no significant difference, and the root length was 53.9 mm, which was 5.4 mm shorter than peat moss (7:3), but survival rate, shoot length, leaf length, and leaf width were different. It was found to be the most suitable soil for the acclimatization of blueberry trees, which grow well in acidic soils, as the growth conditions of the above-ground part were the best and the root measurement results were good (Table 6, Figures 5a, 5b, and 5c). After acclimatizing blueberry plants rooted in-flight for 4 weeks in each soil condition, the appearance of acclimatized seedlings could be confirmed, and it was confirmed that the growth characteristics were superior to other treatments when peat moss was used only (Figure 5d).

Survey items processing area movie
topsoil
(100%) Movie:
peat moss
(5:5) Movie:
peat moss
(7:3) Movie:
peat moss
(3:7) analogy
topsoil
(100%) Analogy:
peat moss
(5:5) Analogy:
peat moss
(7:3) Analogy:
peat moss
(3:7) feet
morse
(100%) Survival rate (%) 100.0 100.0 100.0 96.6 100.0 53.3 33.3 100.0 100.0 number of roots
(dog) average 5.9 7.4 6.7 6.0 6.2 2.2 0.9 6.6 7.2 Deviation 0.7 1.1 0.8 1.7 0.5 0.5 0.8 0.4 2.5 Ieast One 4 One 0 2 0 0 3 3 maximum 12 13 15 11 11 7 4 14 13 root
length
(mm) average 51.7 51.4 59.3 48.2 36.6 15.9 8.5 50.1 53.9 Deviation 2.5 5.2 3.8 4.1 4.3 6.9 8.6 1.8 3.7 Ieast 22 20 3 0 13 0 0 10 13 maximum 86 82 106 83 74 56 46 85 78 shoot
length
(mm) average 46.0 44.3 48.6 55.0 38.3 16.9 8.6 47.7 67.1 Deviation 3.2 4.8 4.5 6.2 3.4 4.4 7.5 3.4 6.1 Ieast 23 20 24 0 20 0 0 21 29 maximum 89 79 69 11.7 91 58 36 75 124 leaf print
(mm) average 7.0 8.2 7.9 8.1 6.6 3.2 1.6 8.5 8.7 Deviation 0.6 0.3 0.6 0.1 0.6 0.8 1.4 0.4 0.7 Ieast 4 5 4 0 5 0 0 4 5 maximum 12 14 12 11 10 9 6 12 12 leaf width
(mm) average 4.6 5.1 5.0 4.7 4.0 2.0 1.0 5.3 5.5 Deviation 0.4 0.2 0.4 0.4 0.5 0.5 0.9 0.3 0.4 Ieast 2 3 3 0 2 0 0 2 3 maximum 7 7 7 6 7 5 4 8 7

Claims (13)

1) Collecting axillary buds where the growth point of the blueberry tree exists, placing the collected buds on Anderson's medium supplemented with zeatin or 2ip to induce shoots and grow;
2) Inducing multiple shoots by planting the grown shoots on Anderson's medium containing a mixture of zeatin and IBA;
3) Inducing rooting by planting the base of the multi-tissue induced blueberry tree on WPM medium containing a mixture of TDZ, IBA, and activated carbon; and
4) Planting the rooting-induced blueberry plants in soil and acclimatizing them in vitro. A method for cultivating blueberry mass propagation, comprising: delete According to clause 1,
The Andersen medium of step 1) is a culture method for mass propagation of blueberry, characterized in that it is a medium prepared by adding 0.5 to 3.0 mg/L of zeatin. According to clause 1,
The Andersen medium of step 1) is a culture method for mass propagation of blueberry, characterized in that it is a medium prepared by single addition of 4.0 to 6.0 mg/L of 2ip. According to clause 1,
The Anderson medium containing a mixture of zeatin and IBA in step 2) is characterized in that it is a medium prepared by adding a mixture of 0.5 to 1.5 mg/L of zeatin and 0.3 to 0.7 mg/L of IBA. Mass propagation culture method. delete delete According to claim 1, the WPM medium to which TDZ, IBA, and activated carbon in step 3) are mixed is added with 0.01 to 1.00 mg/L of TDZ, 0.5 to 2.5 mg/L of IBA, and 0.1 to 2.0 g/L of activated carbon. A method for cultivating mass propagation of blueberry trees, characterized in that it is a manufactured medium. According to clause 1,
The soil in step 4) is a blueberry mass propagation culture method, characterized in that the soil is treated with free topsoil, rich topsoil, or peat moss separately or in combination. According to clause 9,
The above soil is 100% non-fertilized topsoil, non-fertilized topsoil: peat moss (5:5), non-fertilized topsoil: peat moss (7:3), non-fertilized topsoil: peat moss (3:7), 100% fertilized topsoil, fertilized topsoil: peat moss (3: 7) A method of cultivating mass propagation of blueberry trees, characterized in that it is at least one selected from the group consisting of 100% peat moss. The method of claim 10, wherein the soil is 100% peat moss. Blueberry trees produced by the blueberry mass propagation culture method of Paragraph 1. The blueberry tree according to claim 12, wherein the blueberry tree exhibits growth characteristics of 90 to 100% survival rate, 60 to 75 mm shoot length, 7 to 10 mm leaf length, and 5 to 6 mm leaf width.