KR101405390B1 - Method for Plant Formation of Blueberry cv． Bluegold，Eligabeth，Woodard or Tifblue through laminas culture - Google Patents

Abstract

The present invention relates to a medium composition and culture conditions for forming an in-cabbage seedling using the leaf blade culture method of four blueberry varieties, and it requires highly precise techniques such as the harvesting point because it directly uses the leaves of the blueberry seeds cultivated in the cabbage This study was carried out to investigate the method of planting two seedlings of blue hawthorn or bluebird or Elizabeth and two species of rabbit eye strain, Otad and Tiffe Blue, using a leaf culture method which can be relatively easily cultivated. Since the formation rate is high, it is advantageous to increase the number of the plant in the growth culture to expand the culture scale, and since the period of the total plant formation is 8 to 9 months, it is useful as the production technology for expanding and distributing the healthy blueberry domestic tissue culture seedlings Do.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method of forming a plant using a leaf culture method of blueberry, blueberry, elizabeth, orod or teff blue. Bluegold, Eligabeth, Woodard or Tifblue through laminas culture}

The present invention relates to a method for planting blueberry varieties such as Blue Gold, Elizabeth, Otad or Tip Blue.

Blueberries ( Vaccinium spp. ) Is one of the world's top 10 superfoods with a shrubbed crop belonging to the genus Vaccinium ( Cyanoccous ) sections Cyanoccous of the family Ericaceae , anthocyanins, raspberries, etc. It is also known as the 21st Century Fruit Tree because its efficacy has been recognized by its active ingredients. Excellent biocontrol functions include vision recovery and enhancement, retinal degeneration and cataract prevention, carcinogenesis inhibition, and antioxidant effects.

True blueberries (Cyanococcus), a subgenus of these, are found in the eastern United States with many highbush blueberries ( V. corymbosum , in the northeastern United States and Canada Many rabbiteye blueberries (rabbiteye blueberry, V. angustifolium , V. angustifolium , Georgia, etc.) grow in warm areas . ashie . Blueberries, known as the origin of Europe, such as North America and Finland, have been widely known for their fruit flavor and efficacy. Since then, cultivars have been developed since the 1950s and about 200 are known. The species of blueberry that can be cultivated commercially in Korea is high bush species, and the high bush blueberry is mainly divided as follows. The Northern Haibu Clock Blueberry, which grows well in a cold area, Southern Haibu Clock Blueberry which grows well in the cold region, Haibu Clock Blueberry, and Blueberry bloom.

As a high-functioning new income, it has been introduced in Korea since 2000 and the cultivation has started. The total cultivation area in 2011 was 1,082ha (118ha in Chungbuk), 2.1 times more than 2010, . Domestic imports are also increasing every year, and the demand for processed foods such as wine and jam as well as raw materials is surging.

The blueberry seedlings are about 2 ~ 20,000 won per week, which is a high burden on the farm management. In addition, supply of saplings is inadequate compared to demand, and the quality of domestic saplings is lowered, which makes it difficult for cultivated farmers to import saplings and cuttings from China, the US and Japan.

Especially, in the quarantine process, the number of harmful insect pests of imported seedlings is increasing year after year by detecting BlSV (Blueberry scorch virus) from Chinese blueberry seedlings. As a result, there are 177 cases of Peggy 's return measures in 2010. Therefore, there is a need to verify the quality of imported seedlings.

Currently, the Ministry of Agriculture, Forestry and Fisheries is implementing measures to promote the production of disease-free seedlings (2011 ~ 2017) and plans to supply 60% of the demand for main fruit tree seedlings as disease-free seedlings until 2020. to be.

Therefore, as a virus-free disease-free seedlings, it is important to secure the domestic production technology of blueberry tissue culture seedlings, which have strong roots growth, early growth rate, early harvest, fruit quality and tolerance It is important.

Using the Leaf Culture Method The domestic research related to the production of the blueberry tissue culture medium was conducted in 2011 by using the leaflet culture method which was invented and patented by the present inventor, and the method of forming the in-flight vegetation of blueberry varieties such as toro, legacy, or Oregon blue Application No. 10-2011-0138656) is a technology capable of producing tissue culture seeds for three cultivars such as Toro, Legacy and Oregon Blue including cultivation conditions such as the medium composition required for leaf culture.

In addition, domestic related studies for the production of the blueberry varieties related to the present invention for domestic production of the blueberry varieties were carried out in accordance with the inventions of the inventors of blue gold, Elizabeth, Darrow, The method of forming the plant of Orthod or Tiff Blue (Application No. 10-2011-0020374) is the first, and the culture conditions of each kind such as the composition of the medium required for the growth of the growing point include blue gold, Elizabeth, Darrow, It is a technology that can produce tissue culture seeds for varieties.

In order to overcome the limitation of traditional breeding method of blueberry, tissue culture and genetic engineering are being studied. In order to increase the efficiency of the process, the shoots are differentiated from the leaf explant, (Quincy, 2000), as well as tomatoes (Tatchell and Binns, 1986), potatoes (Shddrman and Bevan, 1988) as well as studies on the completion of plants (Billings et al., 1988; Callow et al., 1989) Blackberry (Gupta and Mahalaxmi, 2009) has also been studied.

The culture method that induces shoot formation and plant regeneration using the leaf blade is the ability to reproduce complete plants from a single cell or part of plant tissue by utilizing the totipotency of the plant. Although all the cells have typical performance, the results expressed by the degree of differentiation of the cell tissue, the harvesting site, the composition of the medium, and the culture environment are significantly different. Therefore, it is necessary to optimize the culturing conditions for each crop or variety. In order to induce shoot regeneration from the common leaf, it is necessary to determine the culture conditions that induce the callus formation ability or the direct shoot differentiation ability at the initial stage of the culture since the undifferentiated amorphous cell mass, callus formation, It is most important.

It has been reported that blueberries have a significantly different shoot formation and growth depending on the type and concentration of the growth regulators and additives contained in the culture medium of blueberries (Alexandru F., D. Clapa, and C. Badescu. 2008 ISH Acta Horticulturae 810: IX International Vaccinium Symposium., Sedlak J. and Paprstein F. ISHS Acta Horticulturae 810: IX International Vaccinium Symposium., Pp. 104-109, Jiang YQ, Yu H., Zhang DQ, He SA and Wang CY. IX International Vaccinium Symposium., Clapa D., Fira A., and Rusu T. 2008. Food, Agriculture and Environment 6 (1): 145-147). In addition, even in the same culture conditions, research has been reported that shoot differentiation and plant growth are different depending on the blueberry variety (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) The shoot formation rate and differentiation rate were influenced by the composition of the medium depending on the type and concentration of the additives including the growth regulator and the reaction or specific cultivar to the cytokinin type of each blueberry variety in the plant regeneration process. . Therefore, when using the tissue culture method, it is necessary to optimize the composition of the medium such as the type and concentration of the medium supplement including the growth regulator for each blueberry variety in order to increase the planting rate of blueberry and mass production of successful tissue culture seedlings.

Accordingly, the present inventors formed two plants of blue high-bush type Blue gold or Elizabeth and two species of rabbit eye line, which are excellent in adaptability in Korea and occupy a relatively large area of cultivation, The present inventors have completed the present invention in order to utilize healthy blueberry domestic breakfast cultivated seedlings as source technology for spreading.

The object of the present invention is to provide a blueberry tissue cultured seedlings which can be easily cultivated by using the leaf of the blueberry plant cultivated in the cabin through the leaf blade culture, two kinds of northern highbush blue gold or Elizabeth and rabbit eye line 2 And to provide a method of planting a variety of Otad or Tif Blue.

In order to achieve the above object, the present invention provides a method of planting a blueberry blue gold variety using a lateral culture comprising the steps of:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 to 3 ㎎ / L of Zeatin, 1 to 3 ㎎ / L of Zeatin riboside, 1 to 3 mg / L of ferric sodium salt , 6 to 7 g / L of agar, 20 to 40 g / L of sucrose and 98 to 102 mg / L of myo-inositol, Inducing callus formation and hyperglycemia by culturing 2 days after incubation for 2 weeks by placing the upper surface of the leaf on the medium; And

(b) Zeatin (1 to 3 mg / L), ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, Myo-Inositol 98 to 102 mg / L, Thiamine HCl 1 to 3 mg / L, Nicotinic acid 0.5 to 1.5 mg / L, Pyridoxine HCl 0.5 to 1.5 mg / L, Sucrose 20 to 40 g / L and Agar 5 to 7 g / L, and plant regeneration.

The present invention also provides a method of planting a blueberry Elizabeth variety using leaf blade culture comprising the steps of:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

The present invention also provides a method of planting a blueberry odd variety using a leaf blade culture comprising the steps of:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

In addition, the present invention provides a method of planting a blueberry blue cultivar using leaf blade culture comprising the steps of:

(a) The petiole-free leaves of the inoculated plants were inoculated with 1 to 3 mg / L of zeatin riboside, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) The leaves were plated on a medium supplemented with 98 to 102 mg / L of Myo-Inositol, 20 to 40 g / L of sucrose and 5 to 7 g / L of Agar, Followed by culturing to induce early callus formation and hyperosynthesis; And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

The method of forming the blueberry northern high-bush system two varieties blue gold or two varieties of elizabeth and rabbit eye system according to the present invention is characterized in that the shoot formation ability and the plant regeneration are induced using the leaf blade, Since the leaves of the seedlings are directly used, the seedlings can be cultivated relatively easily without the need of highly precise techniques such as harvesting points, and the number of plantlets is increased And the total period of plant formation is 8 to 9 months, which is useful.

FIG. 1 is a photograph showing the process of plant formation using blue leaf or blueberry, two varieties of blueberry high-bush line, or otad or tiff blue, two varieties of Elizabeth and rabbit eye lines, using the leaf blade culture method according to the present invention.

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.

The present invention used leaves (leaves) of two varieties (blue gold, Elizabeth) and two rabbit eye strains (Otad and Tip blue) of the Northern Highbush line, which are cultivated and propagated in the cabin, as test materials. The cultivation process includes an initial callus formation and a multi-shoot induction step, a plant formation step, a root formation step (rooting step), and an in vitro culture step in which leaves (leaves) of an in- And purifying the plant with the outdoors (greenhouse or packaging).

The present invention provides a method of planting a blueberry blue gold variety using a lateral culture comprising the steps of:

Method for forming inflorescence plants of blueberry blue gold varieties using leaf blade cultures comprising the following steps:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 to 3 ㎎ / L of Zeatin, 1 to 3 ㎎ / L of Zeatin riboside, 1 to 3 mg / L of ferric sodium salt , 6 to 7 g / L of agar, 20 to 40 g / L of sucrose and 98 to 102 mg / L of myo-inositol, Inducing callus formation and hyperglycemia by culturing 2 days after incubation for 2 weeks by placing the upper surface of the leaf on the medium; And

(b) Zeatin (1 to 3 mg / L), ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, Myo-Inositol 98 to 102 mg / L, Thiamine HCl 1 to 3 mg / L, Nicotinic acid 0.5 to 1.5 mg / L, Pyridoxine HCl 0.5 to 1.5 mg / L, Sucrose 20 to 40 g / L and Agar 5 to 7 g / L, and plant regeneration.

In the above method, (c) is a method in which 1 to 2 mg of IBA (indole-3-butyric acid) is added instead of or in addition to 0.1 to 0.3 mg / L of the concentration of ziacin in the additive of (b) / L to induce roots.

The present invention also provides a method of planting a blueberry Elizabeth variety using leaf blade culture comprising the steps of:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

In the above method, (c) is a method in which 1 to 2 mg of IBA (indole-3-butyric acid) is added instead of or in addition to 0.1 to 0.3 mg / L of the concentration of ziacin in the additive of (b) / L to induce roots.

The present invention also provides a method of planting a blueberry odd variety using a leaf blade culture comprising the steps of:

(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

In the above method, (c) is a method in which 1 to 2 mg of IBA (indole-3-butyric acid) is added instead of or in addition to 0.1 to 0.3 mg / L of the concentration of ziacin in the additive of (b) / L to induce roots.

In addition, the present invention provides a method of planting a blueberry blue cultivar using leaf blade culture comprising the steps of:

(a) The petiole-free leaves of the inoculated plants were inoculated with 1 to 3 mg / L of zeatin riboside, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) The leaves were plated on a medium supplemented with 98 to 102 mg / L of Myo-Inositol, 20 to 40 g / L of sucrose and 5 to 7 g / L of Agar, Followed by culturing to induce early callus formation and hyperosynthesis; And

(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.

In the above method, (c) is a method in which 1 to 2 mg of IBA (indole-3-butyric acid) is added instead of or in addition to 0.1 to 0.3 mg / L of the concentration of ziacin in the additive of (b) / L to induce roots.

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

As an inorganic salt of a tissue culture medium other than oxygen, carbon and hydrogen, elements required for plant cell and tissue culture include large amounts of elements such as nitrogen, phosphoric acid, carly, and sulfur and calcium, sodium, magnesium, manganese, molybdenum, , Trace elements such as boron, cobalt and iron are required in a relatively small amount.

These elements play an important role in the development of not only plants but also cells and tissues. Especially, iron (Fe) function is important in trace elements, and plays a role of a cofactor for supplementing and assisting the function of enzymes controlling various metabolic processes occurring in the plant. In particular, there is a layer called lamellae in the chloroplasts in the cells where photosynthetic action of plants occurs. This layer is composed of a protein called cytochrome and contains iron (Fe) in the cytochrome , Which plays an important role in catalyzing photosynthesis, such as the transfer of electrons through oxidation and reduction. It is also known to be essential for plant growth and morphogenesis.

The absorption of iron (Fe) in the culture medium is influenced by the pH. When the pH is higher than 5.2, the water absorption rate is lowered, and iron (Fe) itself does not dissolve in water, . In the case of commercially developed media, Fe was supplied in the form of Fe (SO ₄ ) _3. To overcome the above-mentioned problems, FeSO ₄ 2H ₂ O was added to the medium to prepare ferric sodium salt (FeNa (EDTA) chelate, and the iron (Fe) in the amorphous state can be absorbed even when the pH of the medium is increased to 8, so that the plant can continue to use it.

Zeatin or Zeatin riboside, which is a growth regulator, can be used in combination with other cytotoxic agents such as benzylaminopurine, Thidiazuron, kinetin, and dimethylallyl aminopurine (2iP) It is a plant growth hormone of the cytokinis type and promotes the cell division of the plant. Especially, it is known that it suppresses the growth of the underground part and promotes the growth of the overgrown part. Therefore, it is widely used to propagate and grow shoots in the mass propagation of the plant.

When zitin or zeatin riboside is added at a concentration of 3 mg / L or more at the time of shoot induction, the plant growth is inhibited and the shoot generation is delayed or suppressed and the plant mortality rate is increased And 1 mg / L or less, the callus formation rate is increased and the shoot formation rate is lowered.

When FeNa-EDTA is added at a concentration of 73.4 ㎎ / L or higher, there is a problem that the leaf of the shoot is reddish and the rate of browning or breaking is increased. When added at 27.6 ㎎ / L or less, There is a problem of deterioration. In addition, when Myo-Inositol is added at a concentration of 150 mg / L or more, there is a problem that the rate of transformation is increased during the plant formation process. When the amount is less than 50 mg / L, shoot growth is deteriorated. When thiamine HCl is added at a concentration of 3 mg / L or more, there is a problem that the plant mortality and browning rate are increased. When the concentration is less than 1 mg / L, shoot growth is deteriorated. When nicotinic acid is added at a concentration of more than 1.5 ㎎ / L, there is a problem that the plant mortality and browning rate are increased. When the amount is less than 0.5 mg / L, shoot growth is deteriorated. When Pyridoxine HCl (Pyridoxine HCl) is added at a concentration of 1.5 mg / L or more, there is a problem that the plant mortality and browning rate are increased. When the concentration is less than 0.5 mg / L, shoot growth is deteriorated. In addition, when sucrose is added at a concentration of 50 g / L or more, the sugar concentration in the medium becomes excessively high and the reverse osmosis phenomenon occurs, so that the water inside the plant tissue is likely to escape to the medium, And there is a problem that the plant is damaged such as stem and leaf of the plant are dried in the middle of the growth. When sucrose is added at a concentration of 10 g / L or less, the concentration of carbon and energy-causing sugars in the plant is drastically lowered, which slows growth of shoots and inhibits growth at the later stage of the cultivation step. When agar, which acts to solidify a liquid medium into a solid state, is added at a concentration of 7 g / L or more, the strength (hardness) of the medium becomes excessively high, There is a problem in that it can not be spread out, and when it is added at 5 g / L or less, there is a problem that the plant can not be properly supported on the medium.

The addition of zeatin or zeatin riboside at a concentration of 3 mg / L or more during the hyperhidrosis induces an increase in plant mortality and variability, and the addition of 1 mg / L or less , There is a problem in that the planting rate is lowered and plant growth is inhibited.

The addition of indole-3-butyric acid (IBA) above 4.0 ㎎ / L during roots induction suppresses root formation and causes a higher rate of death such as stem and leaf browning of plant , And when it is added at a concentration of 0.5 mg / L or less, there is a problem that roots are not formed well.

In the present invention, the term " basic medium " means a culture medium containing only the most basic substances necessary for plant cultivation. In the present invention, WPM (Lloyd & McCown Woody Plant Basal Salt Medium) and MS (Murashige & Skoog Medium, Basal Salt mixture, NH ₄ NO ₃ Free) was used as a basic medium. Zeatin or Zeatin riboside, ferric sodium salt-EDTA (FeNa-EDTA), myoinositol The medium is used as a medium for the growth of blueberries by addition of Myo-Inositol, Thiamine HCl, Nicotinic acid, Pyridoxine HCl, Sucrose and Agar. Respectively.

In the present invention, the pH of the basic medium is 3 to 7. When the pH of the medium is higher than 7, the growth of the plant may be delayed because it does not match the characteristic of blueberry which grows well in the acidic state. When the pH is lower than 3, the calcium absorption And calcium deficiency, which can result in necrosis of the growth point. Therefore, it is preferable to adjust the pH of the medium to 3 to 7, more preferably to use the pH of 4.5 to 5.5, but it 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.

< Example  1>

Leaf culture of ' Blue Gold ' varieties

<1-1> Blue Gold 'Varieties of Leaf blade  abstraction

The petiole was removed from the leaves of the blueberry blue gold cultivated in the cabin, and the adaxial side of the leaves was leveled so as to contact with the callus forming medium.

<1-2> Blue Gold 'Varieties of Callus  And Rechincho  Induction culture

Early clallus and mutli-shoot induction media were grown in medium with 1/2 WPM (Lloyd & McCown Woody Plant Basal Salt Medium, MBcell) with growth regulators Zeatin and Giatin Ribo Zeatin ridoside and four additives were added to prepare a medium. The composition of the medium was as shown in Table 1 below.

Each medium was mixed and prepared, and then 80 mL of each culture was dispensed into a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized at 121 ° C for 15 minutes. The leaves (leaves) were streaked in 15 culture dishes and incubated for 2 weeks under dark condition in a culture room maintained at 25 ± 1 ℃. And cultured for about 16 weeks. The initial callus formation and shoot differentiation characteristics of the 18th day of culture were as shown in Table 2 below.

<1-3> Blue Gold 'Growth Culture for Plant Formation of Varieties

After initial callus formation and hyperchromic induction culture for 16-18 weeks after lobule healing, plantlet formation and plant regeneration were induced for 4 to 6 weeks in the culture medium containing the medium of Table 3 below . The regenerated 24-well cultivated plants were characterized as follows [Table 4].

At this time, the plantlet formation and plant regeneration medium in the following [Table 3] were prepared by adding 7 kinds of additives such as growth regulator, zitin, and other additives to the medium in which WPM medium and general MS medium were mixed at a ratio of 3: 1.

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

In order to form roots of the blue gold varieties, 1 mg / L of IBA or 2 mg / L of IBA was added to the medium of which the zeatin concentration was reduced to 0.1-0.3 mg / L or not at all in the medium composition shown in Table 3 below. ㎎ / L was used as a rooting medium and cultured for about 1 month. At this time, the survival rate was improved when the culturing bottle was used for culture of the external air circulation.

At this time, when the total length of the seedling plant was sufficiently grown to 5 cm or more, it was possible to purify the seedlings directly into the outdoors (greenhouse) without roots culturing to form roots.

<1-5> Blue Gold 'Cultivation of cultivated cultivated seedlings for purification process

The roots of roots formed through roots cultivation, or blue gold seedlings which have not yet formed roots but are fully grown at least 5 times, must be purified from the cabin to the outdoors (greenhouse) before they become fully planted seedlings. After planting a mixture of peat moss and perlite in a ratio of 1: 1 (v: v), the blueberry seedlings were planted in pots (inner diameter 110 mm, height 113 mm), 30-40% of sunlight shaded, , A transparent vinyl was covered in a greenhouse maintained at a minimum temperature of 18 ° C, and the curing treatment was performed in a state in which humidity was maintained well. While checking the condition of the seedlings, the plastic was gradually removed and exposed to the atmosphere to completely cure it.

Early callus formation and hypertrophy induction medium composition of blue gold varieties Name
content MgSO ₄ 90.30 mg / L KNO ₃ 950.00 mg / L KH ₂ PO ₄ 85.00 mg / L CaCl ₂ 166.01 mg / L CoCl ₂ 6H ₂ O 0.0125 mg / L ZnSO ₄ 7H ₂ O 4.30 mg / L Na ₂ MoO ₄ 2H ₂ O 0.125 mg / L MnSO ₄ H ₂ O 8.45 mg / L H ₃ BO ₃ 3.10 mg / L KI 0.415 mg / L FeNaEDTA 55.05 mg / L CuSO ₄ 5H ₂ O 0.125 mg / L myo-Inositol 100 mg / L Sucrose 20.00 g / L Zeatin 1.00 mg / L Zeatin Riboside 1.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

18th Parking Callus formation and shoot differentiation characteristics Callus formation rate (%) Differentiation rate of shoots (%) Shincho / plantlet) New plant length (mm) 48.7 ± 3.4 24.7 ± 1.2 24.7 ± 4.0 21.4 ± 2.1

Plant-forming medium composition of blue gold varieties Name
content NH ₄ NO ₃ 300.00 mg / L MgSO ₄ 180.60 mg / L KNO ₃ 475.00 mg / L K ₂ SO ₄ 742.00 mg / L KH ₂ PO ₄ 170.00 mg / L _{Ca (NO 3) 2 4H 2} O 289.50 mg / L CaCl ₂ 137.38 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 20.95 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.2075 mg / L FeNaEDTA 45.875 mg / L Na ₂ EDTA 27.97 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L myo-Inositol 100 mg / L Nicotinic acid 1.00 mg / L Pyridoxine HCl 1.00 mg / L Thiamine HCl 2.00 mg / L Sucrose 30.00 g / L Zeatin 2.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

Characteristics of blueberry blue gold varieties 24 Plant Formation Rate (%) Number of plantlets New plant length (mm) 68.8 ± 3.6 38.6 ± 3.3 28.4 ± 3.2

< Example  2>

Of 'Elizabeth' varieties Leaf blade  Culture method

<2-1> Variety of 'Elizabeth' Leaf blade  abstraction

The petiole was removed from the leaves of the blueberry Elizabeth cultivars cultivated in the cabin, and the adaxial side of the leaves was leveled so as to contact with the callus formation medium.

<2-2> Varieties of 'Elizabeth' Callus  And Rechincho  Induction culture

Early clallus and mutli-shoot induction media were grown in medium with 1/2 WPM (Lloyd & McCown Woody Plant Basal Salt Medium, MBcell) with growth regulators Zeatin and Giatin Ribo The medium was prepared by adding four additives such as Zeatin riboside or Zeatin and TDZ (Thidiazuron). The composition of the medium was as shown in Table 5 below.

Each medium was mixed and prepared, and then 80 mL of each culture was dispensed into a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized at 121 ° C for 15 minutes. The leaves (leaves) were streaked in 15 culture dishes and incubated for 2 weeks under dark condition in a culture room maintained at 25 ± 1 ℃. And cultured for about 16 weeks. The initial callus formation and shoot differentiation characteristics at the 18th day of culture were as shown in Table 6 below.

<2-3> Growth culture for plant formation of 'Elizabeth' varieties

After initial callus formation and hyperchromic induction culture for 16-18 weeks after lobule healing, plantlet formation and plant regeneration were induced for 4-6 weeks in the culture medium containing the medium of Table 7 below . The characteristics of the 24-hour-old seedlings of the regenerated culture were as shown in Table 8 below.

At this time, the plantlet formation and plant regeneration medium in Table 7 below were prepared by adding 7 kinds of additives such as growth regulator, zitin riboside and the other additives to the medium in which WPM medium and general MS medium were mixed at a ratio of 1: 1 Respectively.

<2-4> To induce roots formation of 'Elizabeth' varieties Rooting  culture

In order to form roots of the plants of the Elizabethan variety, the concentration of zeatin riboside was reduced to 0.1-0.3 mg / L in the medium composition of Table 7, Or 2 ㎎ / L was added to the rooting medium and cultured for about 1 month. At this time, the survival rate was improved when the culturing bottle was used for culture of the external air circulation.

At this time, when the total length of the seedling plant was sufficiently grown to 5 cm or more, it was possible to purify the seedlings directly into the outdoors (greenhouse) without roots culturing to form roots.

<2-5> Tissue Culture of 'Elizabeth' Varieties Purification Process for Seedling Formation

The roots of roots were not formed through roots cultivation, but Elizabethan plants that had not yet formed roots but fully grown more than 5 ㎝ had to undergo a purification process from the air (culture room) to the outdoors (greenhouse) to form a complete tissue culture seedling. After planting a mixture of peat moss and perlite in a ratio of 1: 1 (v: v), the blueberry seedlings were planted in pots (inner diameter 110 mm, height 113 mm), 30-40% of sunlight shaded, , A transparent vinyl was covered in a greenhouse maintained at a minimum temperature of 18 ° C, and the curing treatment was performed in a state in which humidity was maintained well. While checking the condition of the seedlings, the plastic was gradually removed and exposed to the atmosphere to completely cure it.

Early callus formation and hypertrophy induction medium composition of Elizabeth varieties Name
content MgSO ₄ 90.30 mg / L KNO ₃ 950.00 mg / L KH ₂ PO ₄ 85.00 mg / L CaCl ₂ 166.01 mg / L CoCl ₂ 6H ₂ O 0.0125 mg / L ZnSO ₄ 7H ₂ O 4.30 mg / L Na ₂ MoO ₄ 2H ₂ O 0.125 mg / L MnSO ₄ H ₂ O 8.45 mg / L H ₃ BO ₃ 3.10 mg / L KI 0.415 mg / L FeNaEDTA 55.05 mg / L CuSO ₄ 5H ₂ O 0.125 mg / L myo-Inositol 100 mg / L Sucrose 20.00 g / L Zeatin 1.00 mg / L Zeatin Riboside 1.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

18th Parking Callus formation and shoot differentiation characteristics Callus formation rate (%) Differentiation rate of shoots (%) Shincho / plantlet) New plant length (mm) 52.6 ± 2.2 25.9 ± 3.1 4.0 ± 0.7 8.1 ± 0.5

Plant-forming medium composition of Elizabeth variety Name
content NH ₄ NO ₃ 200.00 mg / L MgSO ₄ 180.60 mg / L KNO ₃ 950.00 mg / L K ₂ SO ₄ 495.00 mg / L KH ₂ PO ₄ 170.00 mg / L _{Ca (NO 3) 2 4H 2} O 193.00 mg / L CaCl ₂ 202.26 mg / L CoCl ₂ 6H ₂ O 0.0125 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 19.60 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.415 mg / L FeNaEDTA 55.05 mg / L Na ₂ EDTA 18.65 mg / L FeSO ₄ 7H ₂ O 13.92 mg / L CuSO ₄ 5H ₂ O 0.13 mg / L myo-Inositol 100 mg / L Nicotinic acid 1.00 mg / L Pyridoxine HCl 1.00 mg / L Thiamine HCl 2.00 mg / L Sucrose 30.00 g / L Zeatin Riboside 2.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

24 Wed Species Characteristics of Elizabeth Varieties Plant Formation Rate (%) Number of plantlets New plant length (mm) 57.5 ± 3.4 8.6 ± 1.2 24.5 ± 2.0

< Example  3>

' Otad 'Varieties of Leaf blade  Culture method

<3-1> Otad 'Varieties of Leaf blade  abstraction

The petiole was removed from the leaves of the blueberry odad variety cultivated in the cabin, and the adaxial side of the leaves was leveled so as to contact with the callus formation medium.

<3-2> Otad 'Varieties of Callus  And Rechincho  Induction culture

Early clallus and mutli-shoot induction media were grown in media supplemented with Anderson (Anderso Salt Medium, MBcell) medium and normal MS (Murashige & Skoog Medium, Mod. No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) medium was mixed at a ratio of 1: 1. Four different additives were added to the medium in addition to the growth regulators Zeatin riboside and TDZ (Thidiazuron). The composition of the medium was as shown in Table 9 below.

Each medium was mixed and prepared, and then 80 mL of each culture was dispensed into a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized at 121 ° C for 15 minutes. The leaves (leaves) were streaked in 15 culture dishes and incubated for 2 weeks under dark condition in a culture room maintained at 25 ± 1 ℃. And cultured for about 16 weeks. The initial callus formation and shoot differentiation characteristics of the 18th day of culture were as shown in Table 10 below.

<3-3> Otad 'Growth Culture for Plant Formation of Varieties

After initial callus formation and hyperchromic induction culturing for 16-18 weeks after lobule healing, plantlet formation and plant regeneration were induced for 4 to 6 weeks in the culture medium containing the medium of Table 11 below . The characteristics of the 24-hour-old seedlings of the regenerated culture were as shown in Table 12 below.

At this time, the plantlet formation and plant regeneration medium of the following [Table 11] were prepared by adding 7 kinds of additives such as a growth regulator Zeatin riboside to the medium in which WPM medium and general MS medium were mixed at a ratio of 1: 1 To prepare a culture medium.

<3-4> Otad 'For inducing root formation in varieties Rooting  culture

In order to form roots of a plant variety of Otad variety, the concentration of zeatin riboside was reduced to 0.1-0.3 mg / L in the medium composition shown in Table 7 below, or 1 mg / L or 2 ㎎ / L was added to the rooting medium and cultured for about 1 month. At this time, when the culture bottle having a hole in the culture bottle cap is used to circulate the outside air, the survival rate can be increased when the culture is excellently performed.

At this time, when the total length of the seedling plant was sufficiently grown to 5 cm or more, it was possible to purify the seedlings directly into the outdoors (greenhouse) without roots culturing to form roots.

<3-5> Otad 'Cultivation of cultivated cultivated seedlings for purification process

The roots of roots were not formed by roots cultivation or the roots of roots were not formed but the roots of the roots of 5 or more centimeters were fully purified from the cabin to the outside (greenhouse) . After planting a mixture of peat moss and perlite in a ratio of 1: 1 (v: v), the blueberry seedlings were planted in pots (inner diameter 110 mm, height 113 mm), 30-40% of sunlight shaded, , A transparent vinyl was covered in a greenhouse maintained at a minimum temperature of 18 ° C, and the curing treatment was performed in a state in which humidity was maintained well. While checking the condition of the seedlings, the plastic was gradually removed and exposed to the atmosphere to completely cure it.

Early callus formation and hypertrophy induction medium composition of Otad variety Name
content NH ₄ NO ₃ 200.00 mg / L MgSO ₄ 180.62 mg / L KNO ₃ 1190.00 mg / L KH ₂ PO ₄ 85.00 mg / L CaCl ₂ 332.11 mg / L CoCl ₂ 6H ₂ O 0.025 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 16.90 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.565 mg / L FeNaEDTA 55.05 mg / L Na ₂ EDTA 37.25 mg / L FeSO ₄ 7H ₂ O 27.85 mg / L CuSO ₄ 5H ₂ O 0.025 mg / L myo-Inositol 100.00 mg / L Sucrose 20.00 g / L Zeatin Riboside 1.00 mg / L TDZ 1.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

18th Parking Callus formation and shoot differentiation characteristics Callus formation rate (%) Differentiation rate of shoots (%) Shincho / plantlet) New plant length (mm) 25.3 ± 1.1 49.2 ± 4.1 3.5 ± 0.6 10.1 ± 2.2

Plant-forming medium composition of Otad variety Name
content NH ₄ NO ₃ 100.00 mg / L MgSO ₄ 180.60 mg / L KNO ₃ 1425.00 mg / L K ₂ SO ₄ 247.50 mg / L KH ₂ PO ₄ 170.00 mg / L _{Ca (NO 3) 2 4H 2} O 96.50 mg / L CaCl ₂ 267.14 mg / L CoCl ₂ 6H ₂ O 0.01875 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 18.25 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.6225 mg / L FeNaEDTA 64.22 mg / L Na ₂ EDTA 9.32 mg / L FeSO ₄ 7H ₂ O 6.96 mg / L CuSO ₄ 5H ₂ O 0.08 mg / L myo-Inositol 100 mg / L Nicotinic acid 1.00 mg / L Pyridoxine HCl 1.00 mg / L Thiamine HCl 2.00 mg / L Sucrose 30.00 g / L Zeatin Riboside 2.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

24th Issue The characteristics of oil plants of Otad variety Plant Formation Rate (%) Number of plantlets New plant length (mm) 57.5 ± 3.4 8.6 ± 31.2 24.5 ± 2.0

< Example  4> Tiff Blue 'Varieties of Leaf blade  Culture method

<4-1> Tiff Blue 'Varieties of Leaf blade  abstraction

The petiole was removed from the leaves of the blueberry leaf blue cultivated in the cabin, and the adaxial side of the leaves was leveled so as to contact with the callus forming medium.

<4-2> Tiff Blue 'Varieties of Callus  And Rechincho  Induction culture

Early clallus and mutli-shoot induction media were grown in media supplemented with Anderson (Anderso Salt Medium, MBcell) medium and normal MS (Murashige & Skoog Medium, Mod. No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free, Duchefa) medium at a ratio of 1: 1 was prepared by adding the growth regulator Zeatin riboside and four other additives. The composition of the medium was as shown in Table 13 below.

Each medium was mixed and prepared, and then 80 mL of each culture was dispensed into a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized at 121 ° C for 15 minutes. The leaves (leaves) were streaked in 15 culture dishes and incubated for 2 weeks under dark condition in a culture room maintained at 25 ± 1 ℃. And cultured for about 16 weeks. The initial callus formation and shoot differentiation characteristics of the 18th culture were as shown in Table 14 below.

<4-3> Tiff Blue 'Growth Culture for Plant Formation of Varieties

After initial callus formation and hyperchromic induction culturing for 16-18 weeks after lobe formation, plantlet formation and plant regeneration were induced for 4-6 weeks in the culture medium containing the medium of Table 15 below . The characteristics of the 24-hour-old rehydrated culture were as shown in Table 16 below.

At this time, the plantlet formation and plant regeneration medium of the following [Table 15] were prepared by adding 7 kinds of additives such as growth regulator, zitin riboside and the other additives to the mixture of WPM medium and general MS medium at a ratio of 3: 1 Respectively.

<4-4> Tiff Blue 'For inducing root formation in varieties Rooting  culture

In order to form roots of the plant cultivars of the Tiff Blue variety, the concentration of zeatin riboside in the medium composition of Table 15 below was reduced to 0.1-0.3 mg / L, or IBA 1 or 2 ㎎ / L was used as a rooting medium and cultured for about 1 month. At this time, the survival rate was improved when the culturing bottle was used for culture of the external air circulation.

At this time, when the total length of the seedling plant was sufficiently grown to 5 cm or more, it was possible to purify the seedlings directly into the outdoors (greenhouse) without roots culturing to form roots.

<4-5> Tiff Blue 'Cultivation of cultivated cultivated seedlings for purification process

The roots of roots were not formed by roots cultivation, but Ti - bluer plants which had not yet formed roots but fully grown more than 5 ㎝ had to be purified from the inflorescence (culture room) to the outside (greenhouse) . After planting a mixture of peat moss and perlite in a ratio of 1: 1 (v: v), the blueberry seedlings were planted in pots (inner diameter 110 mm, height 113 mm), 30-40% of sunlight shaded, , A transparent vinyl was covered in a greenhouse maintained at a minimum temperature of 18 ° C, and the curing treatment was performed in a state in which humidity was maintained well. While checking the condition of the seedlings, the plastic was gradually removed and exposed to the atmosphere to completely cure it.

Early callus formation and hyperhidrosine induction medium composition of Tiff Blue varieties Name
content NH ₄ NO ₃ 200.00 mg / L MgSO ₄ 180.62 mg / L KNO ₃ 1190.00 mg / L KH ₂ PO ₄ 85.00 mg / L CaCl ₂ 332.11 mg / L CoCl ₂ 6H ₂ O 0.025 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 16.90 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.565 mg / L FeNaEDTA 55.05 mg / L Na ₂ EDTA 37.25 mg / L FeSO ₄ 7H ₂ O 27.85 mg / L CuSO ₄ 5H ₂ O 0.025 mg / L myo-Inositol 100.00 mg / L Sucrose 20.00 g / L Zeatin Riboside 2.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

18th Parking Callus formation and shoot differentiation characteristics Callus formation rate (%) Differentiation rate of shoots (%) Shincho / plantlet) New plant length (mm) 41.7 ± 4.2 19.1 ± 1.7 2.8 ± 0.3 11.8 ± 1.7

Plant-forming medium composition of Tiff Blue variety Name
content NH ₄ NO ₃ 300.00 mg / L MgSO ₄ 180.60 mg / L KNO ₃ 475.00 mg / L K ₂ SO ₄ 742.50 mg / L KH ₂ PO ₄ 170.00 mg / L _{Ca (NO 3) 2 4H 2} O 289.50 mg / L CaCl ₂ 137.38 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L ZnSO ₄ 7H ₂ O 8.60 mg / L Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L MnSO ₄ H ₂ O 20.95 mg / L H ₃ BO ₃ 6.20 mg / L KI 0.2075 mg / L FeNaEDTA 48.875 mg / L Na ₂ EDTA 27.97 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L myo-Inositol 100 mg / L Nicotinic acid 1.00 mg / L Pyridoxine HCl 1.00 mg / L Thiamine HCl 2.00 mg / L Sucrose 30.00 g / L Zeatin Riboside 2.00 mg / L Agar 5.50 g / L The pH of the medium 5.0

Characteristics of 24 plants of blueberry leaf blue varieties Plant Formation Rate (%) Number of plantlets New plant length (mm) 55.9 ± 4.4 11.1 ± 0.3 30.1 ± 1.4

Claims (9)

Method for forming inflorescence plants of Bluegold Bluegold varieties using leaflet cultures comprising the following steps:
(a) petiole-free leaves of in-flight cultivated plants were treated with 1 to 3 ㎎ / L of Zeatin, 1 to 3 ㎎ / L of Zeatin riboside, 1 to 3 mg / L of ferric sodium salt , 6 to 7 g / L of agar, 20 to 40 g / L of sucrose and 98 to 102 mg / L of myo-inositol, Inducing callus formation and hyperglycemia by culturing 2 days after incubation for 2 weeks by placing the upper surface of the leaf on the medium; And
(b) Zeatin (1 to 3 mg / L), ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, Myo-Inositol 98 to 102 mg / L, Thiamine HCl 1 to 3 mg / L, Nicotinic acid 0.5 to 1.5 mg / L, Pyridoxine HCl 0.5 to 1.5 mg / L, Sucrose 20 to 40 g / L and Agar 5 to 7 g / L, and plant regeneration.
Method of Forming Inflorescence Plants of Blueberry Eligabeth Varieties Using Leaf Culture Including the Following Steps:
(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And
(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.
Method for forming inflorescence plants of blueberry orchard varieties using leaf blade cultures comprising the following steps:
(a) petiole-free leaves of in-flight cultivated plants were treated with 1 ~ 3 ㎎ / L of Zeatin, 1 ~ 3 ㎎ / L of Zeatin riboside or TDZ Inositol 98 to 102 mg / L, Sucrose 20 to 40 g / L, and Agar (Lactobacillus amyloliquefaciens) in an amount of 0.5 to 1.5 mg / L of Thidiazuron, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) (Agar) 5 ~ 7 g / L in a medium supplemented with 6 leaves, inducing initial callus formation and hyperosynthesis by incubating for 2 weeks in an incubator for 2 weeks. And
(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.
Method of forming inflorescence in a blueberry Tfblue variety using leaf blade culture comprising the following steps:
(a) The petiole-free leaves of the inoculated plants were inoculated with 1 to 3 mg / L of zeatin riboside, 35 to 37 mg / L of ferric sodium salt (FeNa-EDTA) The leaves were plated on a medium supplemented with 98 to 102 mg / L of Myo-Inositol, 20 to 40 g / L of sucrose and 5 to 7 g / L of Agar, Followed by culturing to induce early callus formation and hyperosynthesis; And
(b) Zeolin riboside 1 to 3 mg / L, ferric sodium salt (FeNa-EDTA) 35 to 37 mg / L, myo-inositol 98 to 102 mg / Thiamine HCl, 0.5 to 1.5 mg / L of Nicotinic acid, 0.5 to 1.5 mg / L of Pyridoxine HCl, 20 to 40 g / L of Sucrose, Formation and plant regeneration of the seedling plant in a medium supplemented with 5 to 7 g / L of 8 seeds.
The method according to any one of claims 1 to 4, further comprising the following step (c):
(c) culturing the hyperhidrosis in a medium containing 0.1 to 0.3 mg / L of zeatin riboside or 1 to 2 mg / L of indole-3-butyric acid (IBA) To induce roots.
2. The method according to claim 1, wherein the medium of step (a) is WPM (Lloyd & McCown Woody Plant Basal Salt Medium) medium and the medium of step (b) & Skoog Medium) medium at a ratio of 3: 1.
The method according to claim 2, wherein the medium of step (a) is WPM medium and the medium of step (b) is medium of WPM medium and MS medium at a ratio of 1: 1 Wherein the method comprises the steps of:
The method according to claim 3, wherein the medium of step (a) is a medium prepared by mixing Anderson (Anderson Salt Medium) medium and general MS medium at a ratio of 1: 1 as a basic medium, Wherein the medium is a mixture of WPM medium and general MS medium at a ratio of 1: 1.
[5] The method of claim 4, wherein the medium of step (a) is a medium in which 1: 1 mixture of Anderson medium and general MS medium is used as a basic medium, and the medium of step (b) Wherein the medium is a mixture of normal MS medium and 3: 1 ratio.

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