KR20120102300A - Method for plant formation of blueberry cv. bluegold, elizabeth, darrow, woodard or tifblue through apical meristem culture - Google Patents

Abstract

PURPOSE: A method for forming Bluegold, Elizabeth, Darrow, Woodard or Tifblue by culturing growth point is provided to form a new shoot without callus formulation and to produce a large amount of blueberry plant of high quality. CONSTITUTION: A method for forming plant of blueberry species, Bluegold comprises a step of placing growth point of the Bluegold in a medium containing 1-3 mg/L of zeatin, 27.6-55.05 mg/L of FeNa-EDTA, 50-150 mg/L of myo-inositol, 1-3 mg/L of thiamine HCl, 0.5-1.5 mg/L of nicotinic acid, 0.5-1.5 mg/L of pyridoxine HCl, 10-50 g/L of sucrose, and 5-7 g of agar to form a new shoot; and a step of culturing the new shoot in a medium containing 1-3 mg/L of zeatin, 36.7-73.4 mg/L of FeNa-EDTA, 50-150 mg/L of myo-inositol, 1-3 mg/L of thiamine, 0.5-1.5 mg/L of nicotinic acid, 0.5-1.5 mg/L of pyridoxine HCl, 10-50 g/L of sucrose, and 5-7 g agar.

Description

Method of plant formation of blue gold varieties, such as blue gold, Elizabeth, Darrow, Ooth or Tiff blue using a growth point culture method. Bluegold, Elizabeth, Darrow, Woodard or Tifblue through apical meristem culture}

The present invention relates to a method of forming plants of blueberry varieties Blue Gold, Elizabeth, Darrow, Ooth or Tif Blue.

Blueberry ( Vaccinium spp.) Is a shrub crop belonging to the family Ericaceae genus Vaccinium section Cyanoccous and is one of the 10 best superfoods in the world. It includes anthocyanins and raspberries. Its utility value is recognized by its active ingredients, and it is also called the 21st century fruit tree. Good bioregulatory functions include restoring and enhancing vision, preventing retinal degeneration and cataracts, inhibiting carcinogenesis, and antioxidant activity.

The hawthorn is divided into four sections as above, and one of the subgenus, true blueberries (Cyanococcus), is found in many highbush blueberries ( V. corymbosum ) in the eastern United States and in the northeastern United States and Canada. It is divided into many lowbush blueberries ( V. angustifolium , Georgia), which grows in warm regions such as rabbiteye blueberries ( V. ashie .) Blueberries, which are native to Europe, including North America and Finland. The taste and efficacy of the fruit became widely known, and since the development of varieties since the 1950's, about 200 species are known now. It is mainly divided into the following: Northern High-Block Blueberry, which grows well in cold regions, Southern High-growth, which grows well in cold regions It is divided into watch blueberry, half-tender high-buoy blueberry, and Levit Eye blueberry.

As a high-functional new income crop, it has been introduced in Korea since 2000, and its cultivation has started. This is concentrated. Domestic imports are also increasing every year, and the demand for processed foods such as wine and jams is rising rapidly as well as raw fruits.

Blueberry seedlings have a big burden in farming management, as seedlings account for a large portion of the initial planting costs of about KRW10,000-20,000 per week. In addition, cultivated farmers are suffering from a lack of seedling supply compared to demand, and the quality of domestic seedlings is poor, so that seedlings and cuttings are imported from China, the United States, and Japan.

In particular, BlSV (Blueberry scorch virus) was detected in Chinese blueberry seedlings during the quarantine process, and has been added to plant quarantine management pests since 2008, increasing the number of cases of disinfecting waste-based transport of imported seedlings.

Therefore, as a virus-free disease-free strain, root production is stronger than cutting seedlings, the initial growth rate is much faster, early harvesting is possible, and the domestic production technology of blueberry tissue culture seedlings is evaluated to be excellent in fruit quality and disease resistance. It is important.

The characteristics of the three varieties of high bushes (Blue Gold, Elizabeth, Darrow) and the two rabbit eye strains (Otad and Tif Blue), which are highly adaptable and occupy relatively large areas, are shown in <Table 1>.

Characteristics of three varieties of blueberry highbush (blue gold, elizabeth, darrow) and rabbit eye 2 varieties (old, tiff blue) Northern Hibu watch case Blue Gold Early spring (early July harvest).
Fruit size is neutral.
USDA registered varieties developed in 1988.
Hybridization of Bluehaven and (Ashworth x Bluecrop).
The height of the tree is about 120 ㎝ and the shape is open.
A lot of flowers and fruits are needed, so you need to work with red flowers or enemies.
Fruit sugar is about 12-14 brix Darlow Late species (harvest late July).
Fruit size is extremely opposite.
USDA registered breed developed in 1965.
(Whareham x Pioneer) and a hybrid of Bluecrop.
The growth of the tree is strong and the effort is about 150 ~ 180cm.
The tree is remodelable.
The color of the fruit is light blue and the fruit is large and the taste is very popular.
The pulp is hard.
The acidity of the fruit is strong, but the flavor is stable because the ratio of sugar acid is stable during the harvest. Elizabeth Midlife breeding (Sea harvest mid-late July).
Fruit size is extremely opposite.
USDA registered breed developed in 1966.
(Katharine x Jersey) and the hybrid of Scammel.
Tree posture is open to upright and mature.
Fruits are large, tasty and excellent in preservation. Rabbit Eye System Tif Blue Mesothelioma (harvest season late July to September).
Fruit size is neutral.
Fostered at Georgia State University in 1955.
USDA registered breed that crossed Ethe and Clara.
It is characterized by strong growth of upright water.
The low temperature required is 600 ~ 650 hours, which is relatively resistant to cold in the rabbit eye system.
Good breed to grow at home.
Water of Brightwell or Powder-blue is required. Otad Mesothelioma (harvest season mid-July to mid-August).
Fruit size is confrontation.
Fostered in Georgia in 1960.
USDA registered breed that crossed Ethel and Callaway.
Until the fruit is ripe, it has a strong sour taste and strong washing.
Low temperature request time is about 350 ~ 400 hours.
Flowers bloom quickly and are weak in the East Sea.

In the case of blueberry tissue culture, studies on the formation and growth of shoots of blueberries differed significantly according to the types and concentrations of growth regulators and additives included in the medium (Alexandru F., D. Clapa, and C. Badescu. 2008 Horticulture. 65 (1): 104-109, Jiang YQ, Yu H., Zhang DQ, He SA and Wang CY ISHS Acta Horticulturae 810: IX International Vaccinium Symposium., Sedlak J. and Paprstein F. ISHS Acta Horticulturae 810: IX International Vaccinium Symposium., Clapa D., Fira A., and Rusu T. 2008. Food, Agriculture and Environment. 6 (1): 145-147). Furthermore, studies have shown that shoot differentiation and plant growth differ between blueberry varieties under the same culture conditions (Gajdosova A., Ostrolucka MG, Libiakova G., Ondruskova E., and Simala D. 2006. Journal of Fruit and Ornamental Plant Research. 14 (Suppl. 1): 103-119, Ostrolucka MG, Libiakova G., Ondruskova E. and Gajdosova A. 2004. Acta Universitatis Latviensis, Biology. 676: 207-212). Therefore, these results indicate that shoot formation and differentiation rate of blueberries depend on the composition of medium and concentration of additives including growth regulators, and the interaction or reaction of each blueberry variety in plant regeneration. the cytokinin type). Therefore, in the case of using the tissue culture method, it is necessary to optimize the composition of the medium such as the type and concentration of medium additives including growth regulators for each blueberry variety in order to increase the plant formation rate of blueberries and to successfully mass-produce tissues.

The present inventors culture the growth point of the blueberry varieties (blue gold, Elizabeth, Darrow, Otard, Tif blue) by omitting the callus formation and immediately organic shoots to form a seedling plant within 5 to 6 months and The present invention was completed by developing culture conditions.

The purpose of the present invention is to skip the callus formation through growth point culture and to minimize the variability rate by immediately eliminating shoot formation and blueberry varieties that can provide a blueberry tissue culture seed in a short time blue gold, Elizabeth, Darrow, Oh It is to provide a method for forming a plant of Ted or Tifblue.

In order to achieve the above object, the present invention provides a method for forming plants of blueberry blue gold varieties using a growth point culture comprising the following steps:

⒜ Blueberry Blue Gold varieties grow from 1 to 3 mg / L Zeatin, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myo-Inositol ), 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L Sucrose And 5 to 7 g of agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar containing; And

신 The shoots were 1 to 3 mg / L Zeatin, 36.7 to 73.4 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myoinositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg Inducing polycythemia by culturing in a medium containing / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

The present invention also provides a method for forming plants of blueberry Elizabeth varieties using growth point culture comprising the following steps:

생 Growth of blueberry Elizabeth varieties 1 ~ 3 mg / L Zetin riboside (Zeatin riboside), 27.6 ~ 55.05 mg / L Ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L Myoinositol (Myo Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without going through a callus forming process; And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In addition, the present invention provides a method for forming a plant of a blueberry Darow variety using a growth point culture comprising the following steps:

생 Growth of blueberry darow varieties 1 ~ 3 mg / L Zetin riboside, 27.6 ~ 55.05 mg / L ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L myoinositol ( Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In addition, the present invention provides a method for forming a plant of a blueberry Otard variety using a growth point culture comprising the following steps:

생 Growing point of blueberry Otard varieties is 1-3 mg / L Zeatin, 27.6-55.05 mg / L Ferricsodium Salt-EDTA, 50-150 mg / L Myo-Inositol ), 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L Sucrose And 5 to 7 g of agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar containing; And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In addition, the present invention provides a method for forming plants of blueberry tip blue varieties using growth point culture comprising the following steps:

⒜ Blueberry Tifblue varieties grow from 1 to 3 mg / L Zein riboside, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L myoinositol Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

Plant formation method of the blueberry varieties according to the present invention blue gold, Elizabeth, Darrow, Otard or Tiff blue is not subjected to the general culturing process to form polycyne after callus formation from the growth point, without the formation of callus By forming, the plant formation process of 10 to 11 months can be shortened to 5 to 6 months. In addition, it does not form a highly variable callus is useful for producing a large amount of blueberry plants of good quality.

1 is a photograph showing the plant formation process using the growth point culture method of blueberry blue gold varieties (A: 10 weeks after growth point, B: 16 weeks, C: 20 weeks, D: 3 months after external purifying E, after 6 months).
Figure 2 is a photograph showing the plant formation process using the growth point culture method of blueberry Elizabeth varieties (A: 10 weeks after growth point, B: 16 weeks, C: 20 weeks, D: 3 months , E: 6 months later).
Figure 3 is a photograph showing the plant formation process using the growth point culture method of blueberry Darow varieties (A: 10 weeks after growth point, B: 16 weeks, C: 20 weeks, D: 3 months E, after 6 months).
Figure 4 is a photograph showing the plant formation process using the growth point culture method of the blueberry Otard variety (A: 10 weeks after growth point, B: 16 weeks, C: 20 weeks, D: 3 months E, after 6 months).
Figure 5 is a photograph showing the plant formation process using the growth point culture method of blueberry Tifblue varieties (A: 10 weeks after growth point, B: 16 weeks, C: 20 weeks, D: 3 months after external purification E, after 6 months).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

This technology uses the blue-berry varieties of three varieties of highbush (blue gold, elizabeth, darrow) and the rabbit eye two varieties (old, tiff blue) to grow the year-end branches of freshly grown eggplants. Early growth point cultivation step (microshoot), taking the growth point (side prominent activity in the stems and roots of the plant and the prominent formation activity such as organ proliferation and organ formation) and collecting it on a solid medium. It consists of a proliferation culture step that actively induces development to speed up plant formation and growth rate, a rooting culture step that induces root formation, and the step of purifying the plant from the cabin (culture room) to the outside (greenhouse or packaging).

In the present invention, the growth point culture is a method of culturing a slice of sperm (or liquid) having several foliar groups, and is a method of cultivating a sperm (or liquid) in which cell division is vigorous for the growth of plants. In the present invention, the growth point of 1 to 2 foliar groups is collected from the solution of the apex of the blueberry branch (side eye that differentiates into leaves in the same year) and then cultured to form a plant of blueberries (FIGS. 1 to 5). .

The present invention provides a plant formation method of blueberry blue gold varieties using growth point culture comprising the following steps:

⒜ Blueberry Blue Gold varieties grow from 1 to 3 mg / L Zeatin, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myo-Inositol ), 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L Sucrose And 5 to 7 g of agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar containing; And

신 The shoots were 1 to 3 mg / L Zeatin, 36.7 to 73.4 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myoinositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg Inducing polycythemia by culturing in a medium containing / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In the above method, 다 the root of the polycincho cultivated in a medium containing 0.1 ~ 0.3 mg / L gelatin or 0.5 ~ 4.0 mg / L indole-3-butyric acid (IBA) It may further comprise the step of inducing.

In the above method, the medium is a medium obtained by mixing WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 3: 1 ratio. It is preferable to use as a basic medium, but is not limited thereto.

The present invention provides a method for forming plants of blueberry Elizabeth varieties using a growth point culture comprising the following steps:

생 Growth of blueberry Elizabeth varieties 1 ~ 3 mg / L Zetin riboside (Zeatin riboside), 27.6 ~ 55.05 mg / L Ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L Myoinositol (Myo Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without going through a callus forming process; And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In the above method, 다 the polycytheria is incubated in a medium containing 0.1 to 0.3 mg / L giatin riboside or 0.5 to 4.0 mg / L indole-3-butyric acid (IBA). It may further comprise the step of inducing roots.

In addition, the medium of the present invention is a medium by mixing WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 1 ratio It is preferable to use as a basic medium, but is not limited thereto.

The present invention provides a plant formation method of the blueberry Darow varieties using a growth point culture comprising the following steps:

생 Growth of blueberry darow varieties 1 ~ 3 mg / L Zetin riboside, 27.6 ~ 55.05 mg / L ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L myoinositol ( Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In the above method, 다 the polycytheria is incubated in a medium containing 0.1 to 0.3 mg / L giatin riboside or 0.5 to 4.0 mg / L indole-3-butyric acid (IBA). It may further comprise the step of inducing roots.

In the above method, the medium of step _iii is prepared by mixing WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 3: 1 ratio. The medium is used as the base medium, and the medium of step 혼합 is mixed with WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 3 ratio. It is preferable to use one medium as a basic medium, but is not limited thereto.

The present invention provides a method for forming a plant of a blueberry Otard variety using a growth point culture comprising the following steps:

생 Growing point of blueberry Otard varieties is 1-3 mg / L Zeatin, 27.6-55.05 mg / L Ferricsodium Salt-EDTA, 50-150 mg / L Myo-Inositol ), 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L Sucrose And 5 to 7 g of agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar containing; And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In the above method, 다 the polycytheria is incubated in a medium containing 0.1 to 0.3 mg / L giatin riboside or 0.5 to 4.0 mg / L indole-3-butyric acid (IBA). It may further comprise the step of inducing roots.

In the above method, the medium of step iii of the present invention is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 1 ratio. The medium of one medium is mixed and the medium of step 는 is 1: 3 using WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free). It is preferable to use one medium as a basic medium by mixing in a ratio, but is not limited thereto.

In addition, the present invention provides a method for forming plants of blueberry tip blue varieties using growth point culture comprising the following steps:

⒜ Blueberry Tifblue varieties grow from 1 to 3 mg / L Zein riboside, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L myoinositol Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And

신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.

In the above method, 다 the polycytheria is incubated in a medium containing 0.1 to 0.3 mg / L giatin riboside or 0.5 to 4.0 mg / L indole-3-butyric acid (IBA). It may further comprise the step of inducing roots.

In the method, the medium is a mixture of WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basic Salt Mixture, NH ₄ NO ₃ Free) in a 3: 1 ratio It is preferable to use as a basic medium, but is not limited thereto.

Tissue culture medium is largely composed of inorganic salts, carbon and energy sources, other organic matter, vitamins, growth regulators, and other additives (agar).

Inorganic salts of tissue culture medium excluding oxygen, carbon, and hydrogen.The elements necessary for plant cell and tissue culture include large elements such as nitrogen, phosphoric acid, kali, and sulfur, calcium, sodium, magnesium, manganese, molybdenum, copper, and zinc. Trace elements required in relatively small quantities, such as, boron, cobalt and iron.

These elements play an important role in the development of cells and tissues as well as plants. In particular, the function of iron (Fe) among the trace elements is important, and serves as a cofactor to complement and assist the function of the enzyme (enzyme) that regulates various metabolic processes occurring in the plant. In particular, there is a layer called lamellar in the chloroplasts in which the photosynthetic action of plants occurs. This layer consists of a protein called cytochrome, and a pigment containing iron (Fe) in the cytochrome. It plays an important role in catalyzing photosynthetic action such as electron transfer function through oxidation and reduction. In addition, it is known to be essential for plant growth and form formation.

Absorption of iron (Fe) in the medium is affected by pH. When the pH is 5.2 or higher, the absorption rate is lowered, and iron (Fe) itself is not easily dissolved in water. . In the past, commercially developed media were fed Fe in the form of Fe (SO ₄ ) ₃ , but in later developed media, FeSO ₄ 2H ₂ O was added to overcome the above-mentioned problems. (chelate) was formed, and this complexed iron (Fe) can be absorbed even when the pH of the medium rises to 8 so that the plant can continue to use it.

Growth regulators, such as giatin or giatin riboside, are plants of the cytokinin family such as benzylaminopurine, thidiazuron, kinetin and dimethylallylaminopurine (2iP). As a growth hormone, it promotes cell division of plants. In particular, it is known to inhibit the growth of the underground and promote the growth of the ground, which is widely used to induce and propagate shoots in the mass reproduction of plants.

In the case of induction of shoots, when the addition of giatin or thiatin riboside to 3 mg / L or more, rather than inhibiting the growth of plants, the development of shoots is delayed or suppressed, the plant mortality is high, below 1 mg / L When added, there is a problem that the callus formation rate is high and shoot formation rate is low. In addition, when ferricsodium salt-EDTA is added at 73.4 mg / L or more, the leaves of shoots turn red and have a high mortality or browning rate, and when added at 27.6 mg / L or less, shoot growth decreases. There is this. And if the addition of more than 150 mg / L myoinositol, there is a problem that the variation rate during the plant formation process, there is a problem that the shoot growth is lowered when added below 50 mg / L. When thiamin HCl is added at 3 mg / L or more, plant mortality and browning rate are increased, and when added at 1 mg / L or less, shoot growth is lowered. In addition, when the nicotinic acid is added at 1.5 mg / L or more, there is a problem in that the plant mortality and browning rate are increased, and when it is added at 0.5 mg / L or less, there is a problem in that shoot growth is lowered. When pyridoxine HCl is added at 1.5 mg / L or more, plant mortality and browning rate are increased, and when added at 0.5 mg / L or less, shoot growth is reduced. In addition, when sucrose is added at 50 g / L or more, the sugar concentration in the medium is excessively increased and reverse osmosis occurs, which increases the possibility of the moisture inside the plant tissues escaping into the medium, and thus the shoots cannot grow normally and grow medium. There is a problem that the plant dies, such as the stem and leaves of the plant to dry. When sucrose is added below 10 g / L, the concentration of sugar and carbon, which is a source of energy of plants, is drastically lowered, which causes slow growth of shoots and inhibits growth at the end of the culturing step. In addition, when the agar, which plays a role in solidifying the liquid medium (gelatinized) and making it into a solid state, is added to 7 g / L or more, the strength (hardness) of the medium is excessively high, so that roots cannot be easily extended from the plant. There is a problem, when added below 5 g / L, there is a problem that the plant is not properly supported in the medium.

In the case of the polycythenic induction, when the gelatin or the gelatin riboside is added at 3 mg / L or more, there is a problem in that the plant mortality and the variability are increased. There is a problem that the lowering and the plant growth is suppressed.

When indole-3-butyric acid is added in an amount of 4.0 mg / L or more during root induction, root formation is rather inhibited and the mortality rate is increased, such as browning of stems and leaves of plants, and 0.5 mg / L or less. When added to, there is a problem that the root formation is not made well.

In the present invention, the "basal medium" means a culture medium containing only the most basic substances necessary for the cultivation of plants. In the present invention, a mixed medium of WPM and MS is used as the basal medium. Tin riboside, ferricsodium salt-EDTA, myoinositol, thiamine HCl, nicotinic acid, pyridoxine HCl, sucrose and agar were further added and used as a medium for the growth point growth of blueberries.

In the present invention, the pH of the basal medium is characterized in that used 3 to 7. If the pH of the medium is used above 7, the growth of the plant may be delayed because it does not match the characteristics of blueberries growing well in acidic condition. If the pH is used below 3, the absorption of calcium (Ca) ion of the plant Inhibition leads to calcium deficiency, which can lead to necrosis of growth points. Therefore, it is preferable to use the pH of the medium adjusted to 3 to 7, and more preferably to use a pH of 4.5 to 5.5, but is not limited thereto.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

‘ Blue Gold ‘Cultivation of growth points of varieties

<1-1> ‘ Blue Gold ‘Growing point of breed

The growth of the blueberry “blue gold” varieties is based on shoot growth (primary branches with freshly grown leaves that year) or shoots in April-May (primary growth) or July-August (secondary growth). The solution was collected from a solution at the apex of the side (side eye that differentiates into leaves in the same year) and used as a culture material. The solution at the apical site was cut out, soaked in 70% ethanol for 30 seconds for surface disinfection, soaked in 3% sodium hypochlorite (NaOCl) solution for 15-20 minutes, and then washed three times for 10 minutes with sterile water. In a clean bench (sterile workbench), 1 to 2 sheets of leaf primordium (a group of cells that originate or produce leaves) are attached to the growth point of 0.3 ~ 0.5 mm in solution using a microscope. Was harvested and cultured.

<1-2> ‘ Blue Gold ‘Breed Shoot ( microshoot Early growth point culture to induce production

Initial growth point culture medium was WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) The medium was prepared by adding growth regulator Ziatin (Zeatin) and 8 additives to the medium in which the medium was mixed in a 3: 1 ratio. Medium composition components are shown in <Table 2>.

Each medium component was prepared by mixing, and then, 10 ml was dispensed into a glass test tube for culture (25 mm in diameter and 150 mm in height), and autoclaved at 121 ° C. for 15 minutes. The growth point was wound one by one in a glass test tube containing medium, and cultured for 1 to 2 months under bright conditions (2,000 ~ 3,000 lux, 16 hours) in a culture room maintained at 25 ± 1 ℃.

<1-3> ‘ Blue Gold ‘Breed Dashincho ( multi - shoot Proliferation Culture to Induce Production

In the initial cultivation process, the growth point of each medium is formed after 1 ~ 2 months incubation period. At this time, it is passaged at 3-4 weeks intervals in the growth medium as shown in <Table 2> to promote growth of shoots. It was. After passing through two or three passages in a new growth medium, shoots grew to some extent, and when multi-shoot was induced, growth of cultures was performed by dividing the base of shoots. When shoots grew during growth and shoot length became 2 cm or more, the culture vessel was changed from a glass test tube (diameter 25 mm, height 150 mm) to a culture bottle (diameter 25 mm, height 150 mm).

<1-4> ‘ Blue Gold ‘For inducing root formation in varieties Rooting  culture

In order to form the roots of the “blue gold” seedlings which have undergone growth culture, IBA 1 or 2 mg was added to the medium without reducing or reducing the concentration of zeatin to 1/10 in the growth culture medium composition of Table 2. / L was added to the rooting medium, and cultured for about 1 month. At this time, there is a hole in the stopper of the culture bottle, so when using a culture bottle in which the outside air is circulated can increase the survival rate during the outside purifying.

When the total length of the "blue gold" seedlings was sufficiently grown to be 5 cm or more, it was possible to directly purify to the outside (greenhouse) without undergoing rooting culture to form roots.

<1-5> ‘ Blue Gold ‘Purification process for the formation of tissue-cultivated seedlings of varieties

Rooted seedlings or roots were not formed through rooting culture, but “Blue Gold” seedlings grown more than 5 cm should be completely incubated in the cabin (culture room) to outside (greenhouse). You will get it. Prepare top soil mixed with peat moss and perlite at 1: 1 (v: v), and then plant blueberry seedlings in pots (internal diameter 110 mm, height 113 mm), shade 30-40% of sunlight In the greenhouse maintained at a minimum of 18 ℃, the transparent vinyl was covered with a hardening treatment in a state where the humidity is well maintained. While checking the condition of the seedlings, the vinyl was slowly rolled out and exposed to the outside air to completely cure.

Culture medium composition of blueberry “blue gold” variety Name content Early Growth Point Culture Medium Proliferation culture medium NH ₄ NO ₃ 300.00 mg / L 300.00 mg / L MgSO ₄ 180.60 mg / L 180.60 mg / L KNO ₃ 475.00 mg / L 475.00 mg / L K ₂ SO ₄ 742.50 mg / L 742.50 mg / L KH ₂ PO ₄ 170.00 mg / L 170.00 mg / L Ca (NO ₃ ) ₂ 4H ₂ O 289.50 mg / L 289.50 mg / L CaCl ₂ 137.38 mg / L 137.38 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L 0.00625 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L 0.25 mg / L MnSO ₄ H ₂ O 20.95 mg / L 20.95 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.2075 mg / L 0.2075 mg / L FeNaEDTA 36.70 mg / L 45.875 mg / L Na ₂ EDTA 27.97 mg / L 27.97 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L 20.88 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L 0.19 mg / L myo-Inositol 100 mg / L 100 mg / L Nicotinic acid 1.00 mg / L 1.00 mg / L Pyridoxine HCl 1.00 mg / L 1.00 mg / L Thiamine HCl 2.00 mg / L 2.00 mg / L Sucrose 30 g / L 30 g / L Zeatin 2.00 mg / L 2.00 mg / L Agar 6 g / L 6 g / L PH of medium 5.0 5.0

Growth method of 'Elizabeth' variety

<2-1> Growth Points of 'Elizabeth' Varieties

The growth method of blueberry “Elizabeth” varieties is the same as that of the 'blue gold' varieties.

<2-2> of the Elizabethan cultivar Shoot ( microshoot Early growth point culture to induce production

Initial growth point culture medium was WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) The medium was prepared by adding the growth regulator zetin riboside and eight additives to the medium in which the medium was mixed in a 1: 1 ratio. The medium composition component is shown in <Table 3>.

Each medium component was prepared by mixing, and then, 10 ml was dispensed into a glass test tube for culture (25 mm in diameter and 150 mm in height), and autoclaved at 121 ° C. for 15 minutes. The growth point was wound one by one in a glass test tube containing medium, and cultured for 1 to 2 months under bright conditions (2,000 ~ 3,000 lux, 16 hours) in a culture room maintained at 25 ± 1 ℃.

<2-3> Of the Elizabethan cultivar Dashincho ( multi - shoot Proliferation Culture to Induce Production

In the initial cultivation process, the growth point of each medium is formed after 1 ~ 2 months of incubation period. At this time, it is passaged every 3 to 4 weeks in the growth medium as shown in <Table 3> to promote growth of shoots. It was. After passing through two or three passages in a new growth medium, shoots grow to some extent, and when multi-shoot is induced, growth of cultures was performed by dividing the base of shoots. When shoots grew during growth and shoot length became 2 cm or more, the culture vessel was changed from a glass test tube (diameter 25 mm, height 150 mm) to a culture bottle (diameter 25 mm, height 150 mm).

<2-4> Induction of Root Formation in 'Elizabeth' Varieties Rooting  culture

In order to form the roots of the "Elizabeth" seedlings which have undergone growth culture, the concentration of zeat riboside in the growth culture medium composition of Table 3 is reduced to 1/10 or IBA 1 or 2 mg / L was added and used as a rooting medium, and cultured for about 1 month. At this time, there is a hole in the stopper of the culture bottle, so when using a culture bottle in which the outside air is circulated can increase the survival rate during the outside purifying.

When the total length of the "Elizabeth" seedlings was sufficiently grown to 5 cm or more, it was possible to immediately purify the greenhouse without going through the rooting culture to form the roots.

<2-5> Purification Process for Forming Tissue Culture Seedlings of 'Elizabeth' Varieties

Rooted seedlings or roots were not formed through rooting cultivation, but “Elizabeth” seedlings grown more than 5 cm in order to form a complete tissue culture seedling only after the process of cultivation from the cabin (culture room) to the outside (greenhouse). do. Purifying from the outside is the same as the 'blue gold' varieties above.

Culture medium composition of blueberry “Elizabeth” varieties Name content Early Growth Point Culture Medium Proliferation culture medium NH ₄ NO ₃ 200.00 mg / L 200.00 mg / L MgSO ₄ 180.60 mg / L 180.60 mg / L KNO ₃ 950.00 mg / L 950.00 mg / L K ₂ SO ₄ 495.00 mg / L 495.00 mg / L KH ₂ PO ₄ 170.00 mg / L 170.00 mg / L Ca (NO ₃ ) ₂ 4H ₂ O 193.00 mg / L 193.00 mg / L CaCl ₂ 202.26 mg / L 202.26 mg / L CoCl ₂ 6H ₂ O 0.0125 mg / L 0.0125 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L 0.25 mg / L MnSO ₄ H ₂ O 19.60 mg / L 19.60 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.415 mg / L 0.415 mg / L FeNaEDTA 36.70 mg / L 55.05 mg / L Na ₂ EDTA 18.65 mg / L 18.65 mg / L FeSO ₄ 7H ₂ O 13.92 mg / L 13.92 mg / L CuSO ₄ 5H ₂ O 0.13 mg / L 0.13 mg / L myo-Inositol 100 mg / L 100 mg / L Nicotinic acid 1.00 mg / L 1.00 mg / L Pyridoxine HCl 1.00 mg / L 1.00 mg / L Thiamine HCl 2.00 mg / L 2.00 mg / L Sucrose 30 g / L 30 g / L Zeatin riboside 2.00 mg / L 2.00 mg / L Agar 6 g / L 6 g / L PH of medium 5.0 5.0

‘ Darlow ‘Cultivation of growth points of varieties

<3-1> ‘ Darlow ‘Growing point of breed

The growth method of blueberry “Darrow” varieties is the same as that of the 'blue gold' varieties.

<3-2> ‘ Darlow ‘Breed Shoot ( microshoot Early growth point culture to induce production

Initial growth point culture medium was WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) To the medium in which the medium was mixed in a 3: 1 ratio, a growth regulator Zetin riboside and eight other additives were added to prepare a medium. The medium composition component is shown in <Table 4>.

Each medium component was prepared by mixing, 10 ml each of the culture glass test tubes (diameter 25 mm, height 150 mm) was used and autoclaved at 121 ° C. for 15 minutes. The growth point was wound one by one in a glass test tube containing medium, and cultured for 1 to 2 months under bright conditions (2,000 ~ 3,000 lux, 16 hours) in a culture room maintained at 25 ± 1 ℃.

<3-3> ‘ Darlow ‘Breed Dashincho ( multi - shoot Proliferation Culture to Induce Production

In the initial cultivation process, the growth point of each medium was formed after 1 ~ 2 months of incubation period, at which time it was passaged to the growth culture medium at intervals of 3 to 4 weeks to promote growth of shoots.

Proliferation culture media were WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) Was added to the medium mixed with a 1: 3 ratio to the growth regulator Zetin riboside (Zeatin riboside) and the addition of eight additives to prepare a medium, the components are shown in Table 4.

After passing through two or three passages in a new growth medium, shoots grew to some extent, and when multi-shoot was induced, growth of cultures was performed by dividing the base of shoots. When shoots grew during growth and shoot length became 2 cm or more, the culture vessel was changed from a glass test tube (diameter 25 mm, height 150 mm) to a culture bottle (diameter 25 mm, height 150 mm).

<3-4> ‘ Darlow Root culture to induce root formation of varieties

In order to form roots of “Darrow” seedlings which have undergone growth culture, the concentration of zeat riboside in the growth culture medium composition of Table 4 is reduced to 1/10 or IBA 1 was added to the medium without addition. Or 2 mg / L was added to use as a rooting medium, and cultured for about 1 month. At this time, there is a hole in the stopper of the culture bottle, so when using a culture bottle in which the outside air is circulated can increase the survival rate during the outside purifying.

When the total length of the "Darrow" seedlings was sufficiently grown to be 5 cm or more, it was possible to immediately purify the air (greenhouse) without going through the rooting culture to form the roots.

<3-5> ‘ Darlow ‘Purification process for the formation of tissue-cultivated seedlings of varieties

Rooted seedlings or roots were not formed through rooting cultivation, but “Darrow” seedlings grown more than 5 cm must be fully cultured in the incubation room to the outside (greenhouse). You will have Purifying from the outside is the same as the 'blue gold' varieties above.

Culture Medium Composition of Blueberry “Darrow” Varieties Name content Early Growth Point Culture Medium Proliferation culture medium NH ₄ NO ₃ 300.00 mg / L 100.00 mg / L MgSO ₄ 180.60 mg / L 180.60 mg / L KNO ₃ 475.00 mg / L 1425.00 mg / L K ₂ SO ₄ 742.50 mg / L 247.50 mg / L KH ₂ PO ₄ 170.00 mg / L 170.00 mg / L Ca (NO ₃ ) ₂ 4H ₂ O 289.50 mg / L 96.50 mg / L CaCl ₂ 137.38 mg / L 267.14 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L 0.01875 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L 0.25 mg / L MnSO ₄ H ₂ O 20.95 mg / L 18.25 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.2075 mg / L 0.6225 mg / L FeNaEDTA 36.70 mg / L 64.22 mg / L Na ₂ EDTA 27.97 mg / L 9.32 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L 6.96 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L 0.08 mg / L myo-Inositol 100 mg / L 100 mg / L Nicotinic acid 1.00 mg / L 1.00 mg / L Pyridoxine HCl 1.00 mg / L 1.00 mg / L Thiamine HCl 2.00 mg / L 2.00 mg / L Sucrose 30 g / L 30 g / L Zeatin riboside 2.00 mg / L 2.00 mg / L Agar 6 g / L 6 g / L PH of medium 5.0 5.0

‘ Otad ‘Cultivation of growth points of varieties

<4-1> ‘ Otad ‘Growing point of breed

The growth method of blueberry “Oodd” varieties is the same as that of the 'blue gold' varieties above.

<4-2> ‘ Otad ‘Breed Shoot ( microshoot Early growth point culture to induce production

Initial growth point culture medium was WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) The medium was prepared by adding growth regulator Ziatin (Zeatin) and 8 additives to the medium in which the medium was mixed in a 1: 1 ratio. The medium composition component is shown in <Table 5>.

Each medium component was prepared by mixing, and then, 10 ml was dispensed into a glass test tube for culture (25 mm in diameter and 150 mm in height), and autoclaved at 121 ° C. for 15 minutes. The growth point was wound one by one in a glass test tube containing medium, and cultured for 1 to 2 months under bright conditions (2,000 ~ 3,000 lux, 16 hours) in a culture room maintained at 25 ± 1 ℃.

<4-3> ‘ Otad ‘Breed Dashincho ( multi - shoot Proliferation Culture to Induce Production

In the initial cultivation process, the growth point of each medium was formed after 1 ~ 2 months of incubation period. At this time, it was passaged to the growth culture medium at intervals of 3 to 4 weeks to promote growth of shoots.

Proliferation culture media were WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) Was added to the medium mixed with a 1: 3 ratio growth medium, Zein riboside (Zeatin riboside) and the addition of eight additives to prepare a medium, the components are shown in Table 5.

After passing through two or three passages in a new growth medium, shoots grew to some extent, and when multi-shoot was induced, growth of cultures was performed by dividing the base of shoots. When shoots grew during growth and shoot length became 2 cm or more, the culture vessel was changed from a glass test tube (diameter 25 mm, height 150 mm) to a culture bottle (diameter 25 mm, height 150 mm).

<4-4> ‘ Otad ‘For inducing root formation in varieties Rooting  culture

In order to form the roots of “Oodd” seedlings which have undergone growth culture, the concentration of zeat riboside in the growth culture medium composition of Table 5 is reduced to 1/10 or IBA 1 was added to the medium without addition. Alternatively, 2 mg / L was added and used as a rooting medium, and cultured for about 1 month. At this time, there is a hole in the stopper of the culture bottle, so when using a culture bottle in which the outside air is circulated can increase the survival rate during the outside purifying.

When the total length of the “Odard” seedlings was sufficiently grown to 5 cm or more, it was possible to immediately purify the air (greenhouse) without going through the rooting culture to form roots.

<4-5> ‘ Otad ‘Purification process for the formation of tissue-cultivated seedlings of varieties

Rooted seedlings or roots were not formed through rooting cultivation, but “Odard” seedlings grown more than 5 cm should be completely incubated in the cabin (culture room) to outside (greenhouse). You will get it. Purifying from the outside is the same as the 'blue gold' varieties above.

Culture Medium Composition of Blueberry “Otard” Varieties Name content Early Growth Point Culture Medium Proliferation culture medium NH ₄ NO ₃ 200.00 mg / L 100.00 mg / L MgSO ₄ 180.60 mg / L 180.60 mg / L KNO ₃ 950.00 mg / L 1425.00 mg / L K ₂ SO ₄ 495.00 mg / L 247.50 mg / L KH ₂ PO ₄ 170.00 mg / L 170.00 mg / L Ca (NO ₃ ) ₂ 4H ₂ O 193.00 mg / L 96.50 mg / L CaCl ₂ 202.26 mg / L 267.14 mg / L CoCl ₂ 6H ₂ O 0.0125 mg / L 0.01875 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L 0.25 mg / L MnSO ₄ H ₂ O 19.60 mg / L 18.25 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.415 mg / L 0.6225 mg / L FeNaEDTA 36.70 mg / L 64.22 mg / L Na ₂ EDTA 18.65 mg / L 9.32 mg / L FeSO ₄ 7H ₂ O 13.92 mg / L 6.96 mg / L CuSO ₄ 5H ₂ O 0.13 mg / L 0.08 mg / L myo-Inositol 100 mg / L 100 mg / L Nicotinic acid 1.00 mg / L 1.00 mg / L Pyridoxine HCl 1.00 mg / L 1.00 mg / L Thiamine HCl 2.00 mg / L 2.00 mg / L Sucrose 30 g / L 30 g / L Zeatin 2.00 mg / L 0.00 mg / L Zeatin riboside 0.00 mg / L 2.00 mg / L Agar 6 g / L 6 g / L PH of medium 5.0 5.0

‘ Tif Blue ‘Cultivation of growth points of varieties

<5-1> ‘ Tif Blue ‘Growth point of breed

The growth method of blueberry “tifblue” varieties is the same as that of the 'blue gold' varieties above.

<5-2> ‘ Tif Blue ‘Breed Shoot ( microshoot Early growth point culture to induce production

Initial growth point culture medium was WPM (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins, MBcell) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) To the medium in which the medium was mixed in a 3: 1 ratio, a growth regulator Zetin riboside and eight other additives were added to prepare a medium. The medium composition component is shown in <Table 6>.

Each medium component was prepared by mixing, and then, 10 ml was dispensed into a glass test tube for culture (25 mm in diameter and 150 mm in height), and autoclaved at 121 ° C. for 15 minutes. The growth point was wound one by one in a glass test tube containing medium, and cultured for 1 to 2 months under bright conditions (2,000 ~ 3,000 lux, 16 hours) in a culture room maintained at 25 ± 1 ℃.

<5-3> ‘ Tif Blue ‘Breed Dashincho ( multi - shoot Proliferation Culture to Induce Production

In the initial cultivation process, the growth point of each medium is formed after 1 ~ 2 months of incubation period. At this time, it is passaged at 3-4 week intervals in the growth medium as shown in <Table 6> to promote growth of shoots. It was. After passing through two or three passages in a new growth medium, shoots grew to some extent, and when multi-shoot was induced, growth of the shoots was performed by dividing the base of shoots. When shoots grew during growth and shoot length became 2 cm or more, the culture vessel was changed from a glass test tube (diameter 25 mm, height 150 mm) to a culture bottle (diameter 25 mm, height 150 mm).

<5-4> ‘ Tif Blue ‘For inducing root formation in varieties Rooting  culture

In order to form the roots of the “tifblue” seedlings grown through the growth culture, the concentration of zeat riboside in the growth culture medium composition of Table 6 was reduced to 1/10 or IBA 1 was added to the medium without addition. Alternatively, 2 mg / L was added and used as a rooting medium, and cultured for about 1 month. At this time, there is a hole in the stopper of the culture bottle, so when using a culture bottle in which the outside air is circulated can increase the survival rate during the outside purifying.

When the total length of the "tifblue" seedlings was sufficiently grown to 5 cm or more, it was possible to immediately purify the air (greenhouse) without going through the rooting culture to form the roots.

<5-5> ‘ Tif Blue ‘Purification process for the formation of tissue-cultivated seedlings of varieties

Rooted seedlings or roots were not formed through rooting cultivation, but “tefblue” seedlings grown more than 5 cm must be completely cultured in the incubation room to the outside (greenhouse). You will have Purifying in the air is the same as the method of the above 'blue gold' breed.

Culture medium composition of blueberry “Tiffblue” varieties Name content Early Growth Point Culture Medium Proliferation culture medium NH ₄ NO ₃ 300.00 mg / L 300.00 mg / L MgSO ₄ 180.60 mg / L 180.60 mg / L KNO ₃ 475.00 mg / L 475.00 mg / L K ₂ SO ₄ 742.50 mg / L 742.50 mg / L KH ₂ PO ₄ 170.00 mg / L 170.00 mg / L Ca (NO ₃ ) ₂ 4H ₂ O 289.50 mg / L 289.50 mg / L CaCl ₂ 137.38 mg / L 137.38 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L 0.00625 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L 0.25 mg / L MnSO ₄ H ₂ O 20.95 mg / L 20.95 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.2075 mg / L 0.2075 mg / L FeNaEDTA 36.70 mg / L 45.875 mg / L Na ₂ EDTA 27.97 mg / L 27.97 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L 20.88 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L 0.19 mg / L myo-Inositol 100 mg / L 100 mg / L Nicotinic acid 1.00 mg / L 1.00 mg / L Pyridoxine HCl 1.00 mg / L 1.00 mg / L Thiamine HCl 2.00 mg / L 2.00 mg / L Sucrose 30 g / L 30 g / L Zeatin riboside 2.00 mg / L 2.00 mg / L Agar 6 g / L 6 g / L PH of medium 5.0 5.0

The present invention can be utilized as a source technology for the expansion and spread of healthy blueberry domestic tissue culture seedlings, to meet the domestic demand of the rapidly growing blueberry seedlings and to obtain the effect of replacing the import of seedlings from foreign countries, high-quality domestic bottled wine ( The spread of virus-free seedlings) and the supply and demand of seedlings will be stabilized. In addition, the mass production of virus-free seedlings could expand the possibility of foreign exports.

Claims (11)

Plant formation method of blueberry blue gold varieties using growth point culture comprising the following steps:
⒜ Blueberry Bluegold varieties grow from 1 to 3 mg / L Zeatin, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myoinositol Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And
신 The shoots were 1 to 3 mg / L Zeatin, 36.7 to 73.4 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L Myoinositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg Inducing polycythemia by culturing in a medium containing / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.
Method of plant formation of blueberry Elizabethi varieties using growth point culture comprising the following steps:
생 Growth of blueberry Elizabeth varieties 1 ~ 3 mg / L Zetin riboside (Zeatin riboside), 27.6 ~ 55.05 mg / L Ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L Myoinositol (Myo Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without going through a callus forming process; And
신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.
Plant Formation Method of Blueberry Darrow Varieties Using Growth Point Cultures Comprising the Following Steps:
생 Growth of blueberry darow varieties 1 ~ 3 mg / L Zetin riboside, 27.6 ~ 55.05 mg / L ferricsodium salt-EDTA (FeNa-EDTA), 50 ~ 150 mg / L myoinositol ( Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g agar (Agar) is dentured to form a shoot without undergoing callus formation process by agar (Agar); And
신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.
Plant formation method of blueberry Woodard varieties using growth point culture comprising the following steps:
생 Growing point of blueberry Otard varieties is 1-3 mg / L Zeatin, 27.6-55.05 mg / L Ferricsodium Salt-EDTA, 50-150 mg / L Myo-Inositol ), 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L Sucrose And 5 to 7 g of agar (Agar) on the medium to form a shoot without undergoing callus formation process; And
신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.
Plant formation method of blueberry Tifblue varieties using growth point culture comprising the following steps:
⒜ Blueberry Tifblue varieties grow from 1 to 3 mg / L Zein riboside, 27.6 to 55.05 mg / L Ferricsodium Salt-EDTA, 50 to 150 mg / L myoinositol Myo-Inositol, 1 to 3 mg / L Thiamine HCl, 0.5 to 1.5 mg / L Nicotinic acid, 0.5 to 1.5 mg / L Pyridoxine HCl, 10 to 50 g / L sucrose (Sucrose) and 5 to 7 g of Agar (Agar) is dentured to form a shoot without going through a callus forming process; And
신 the shoots were 1 to 3 mg / L Zetin riboside, 36.7 to 73.4 mg / L ferricsodium salt-EDTA, 50 to 150 mg / L myoinositol, 1 to 3 mg / L thiamine HCl, 0.5 Inducing polycythemia by culturing in a medium containing ˜1.5 mg / L nicotinic acid, 0.5-1.5 mg / L pyridoxine HCl, 10-50 g / L sucrose, and 5-7 g agar.
The method of claim 1, further comprising the following step iii:
배양 inducing the roots by culturing the polycytheria in a medium containing 0.1-0.3 mg / L giatin or 0.5-4.0 mg / L indole-3-butyric acid (IBA).
The plant forming method according to any one of claims 2 to 5, further comprising the following step iii:
다 to induce roots by culturing the polycytheria in a medium containing 0.1 to 0.3 mg / L giatin riboside or 0.5 to 4.0 mg / L indole-3-butyric acid (IBA) step.
According to claim 1 or 5, wherein the medium is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 3: 1 ratio Plant forming method characterized in that the mixed medium as a base medium.
The medium of claim 2, wherein the medium is a mixture of WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 1 ratio. Plant formation method characterized in that the base medium.
The method of claim 3, wherein the medium of step _iii is mixed with WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 3: 1 ratio One medium is used as the base medium, and the stage 단계 medium is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 3 ratio. Plant forming method characterized in that the mixed medium as a base medium.
The medium of step 4 is a mixture of WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 1 ratio. One medium is used as the base medium, and the stage 단계 medium is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium) and MS medium (Murashige & Skoog Medium, Basal Salt Mixture, NH ₄ NO ₃ Free) in a 1: 3 ratio. Plant forming method characterized in that the mixed medium as a base medium.

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