KR20130071254A - Method for plantlet formation of blueberry cv. toro, legacy or oregonblue using leaf explant culture - Google Patents

Abstract

PURPOSE: A method for plantlet formation of blueberry cv. toro, legacy or oregonblue is provided to increase the number of plantlets during a growth culturing process since a multi-shoot formation rate is higher than a meristem-tip culture. CONSTITUTION: A blueberry leaf is taken into a medium(1) to induce an initial callus and multi-shoot. 1 to 3 mg/L of Zeatin Riboside, 30 to 60 mg/L of FeNa-EDTA, 50 to 150 mg/L of myo-inositol, 1 to 3 mg/L Thiamine HCl, 0.5 to 1.5 mg/L of Nicotinic acid, 0.5 to 1.5 mg/L of Pyridoxine HCl, 10 to 30 g/L of sucrose, and 4 to 8 g/L of agar are added to the medium and then cultured in a next medium(2) to form a plant body. In the next medium, Zeatin Riboside is added at 1/10 of the above concentration or not added, or the medium(2) is cultured in a medium added with 0.1 to 3 mg/L of IBA(Indole-3-Butyric Acid) to induce formation of rood by circulating from a culturing chamber to a greenhouse. The medium(1) includes 1 to 3 mg/L of zeatin, 30 to 60 mg/L of FeNa-EDTA, 50 to 150 mg/L of myo-inositol, 10 to 30 g/L of sucrose, and 4 to 8 g/L of agar. [Reference numerals] (AA) 14 weeks; (BB) 18 weeks; (CC) 24 weeks; (DD) 34 weeks; (EE) 3 months

Description

In-plant seedling formation method of Toro, Legacy, or Oregon blue, a blueberry variety using the leaf culture method {Method for plantlet formation of blueberry cv. Toro, Legacy or Oregonblue using leaf explant culture}

The present invention relates to a method of forming an in-plant seedling of Toro, Legacy or Oregon Blue, which is a blueberry variety using the leaf culture method.

Blueberry ( Vaccinium spp. ) Is a shrub crop belonging to the genus Vaccinium of the family Ericaceae and is one of the top 10 superfoods selected by Time magazine. It is also known as the 21st century fruit tree. Excellent biocontrol functions include vision recovery and enhancement, retinal degeneration and cataract prevention, carcinogenesis inhibition, and antioxidant effects. 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, the supply and demand of seedlings is increasing, such as the lack of seedling supply and the quality of domestic seedlings are poor, so that seedlings and cuttings are imported from China, the United States, and Japan. Especially during quarantine, BlSV ( Blueberry scorch) from Chinese blueberry seedlings The number of harmful pests found in imported seedlings is increasing year by year. As a result, there were 177 cases of disposal and return measures in 2010, raising the need for quality verification of imported seedlings. In order to solve this situation, the Ministry of Food, Agriculture, Forestry and Fisheries is currently implementing measures to supply and sell disease-free seedlings (2011-2017), and by 2020, 60% of the demand for major fruit tree seedlings will be supplied as disease-free seedlings. Development of production technology is a very important point.

As a disease-free strain without viruses, blueberry's tissue culture seed production technology has been evaluated to be more robust in root and tree growth than in cutting seedlings, and its early growth rate is not only possible, but also its fruit quality and disease resistance are excellent. It is necessary to conduct research to establish. In order to overcome the limitations of the traditional breeding methods of blueberries, researches on tissue culture and genetic engineering have been conducted. In order to increase the efficiency of this process, shoots from leaf explants are taken. After the study of differentiating the complete plant (Billings et al., 1988; Callow et al., 1989), it was not only tomatoes (Tatchell and Binns, 1986), but also potatoes (Shddrman and Bevan, 1988), as well as cranberries (Qu, (2000).

The culture method that induces shoot formation and plant regeneration using leaflets is to take advantage of the plant's totipotency, the ability to regenerate the complete plant from a single cell or plant tissue. Although all cells have typical properties, the results expressed by the degree of differentiation of the tissue, the collection site, the composition of the medium, the culture environment, etc. are remarkably different. Therefore, it is necessary to optimize the culture conditions for each crop or variety. In order to induce shoot regeneration from leaflets, the shoots are formed after the formation of callus, which is an undifferentiated irregular cell mass, and thus the culture that induces a strong callus formation ability or direct shoot differentiation ability at the beginning of the culture. Finding the condition is the most important.

Development of lobe cultivation technology for blueberry varieties can increase the efficiency of in-flight cultivation and production systems, accelerate the mass production and production, meet the domestic demand for rapidly growing blueberry seedlings, and import seedlings from foreign countries. It can have a replacement effect. In addition, it will be possible to expand the supply of disease-free stocks without high-quality domestic viruses and stabilize supply and demand of seedlings, and to expand the possibility of overseas export by mass production of seedlings without viruses.

Therefore, the present inventors are trying to establish a technology for producing tissue culture seedlings of blueberries, and are highly adaptable and occupy a relatively large cultivation area, two varieties of Hibush type (Toro, Legacy) and one rabbit eye type (Oregon Blue). Development of the culture and culture conditions for forming seedlings within 8 to 9 months in the plane by culturing the leaflets, and completed the present invention by confirming that it can be used as a source technology for expanding and disseminating blueberry domestic tissue culture seedlings It was.

An object of the present invention is to provide a method for forming plants of Toro, Legacy or Oregon Blue, which is a blueberry variety using the leaf culture method.

In order to solve the above problems, the present invention provides a method for forming plants of blueberry varieties using the leaf culture method comprising the following steps.

1) Blueberry leaves (leaflets) 1 to 3 mg / L Zeatin, FeNa-EDTA 30 to 60 mg / L, Myo-Inositol 50 to 150 mg / L, sucrose Inducing initial callus and polycytheria by flocculation in a medium containing 10 to 30 g / L and 4 to 8 g / L of agar;

2) Zeatin Riboside 1 to 3 mg / L, FeNa-EDTA 30 to 60 mg / L, Myoinositol 50 to 150 mg / L, Thiamine HCl (Thiamine HCl) 1 in the medium of step 1) To 3 mg / L, nicotinic acid 0.5 to 1.5 mg / L, pyridoxine HCl 0.5 to 1.5 mg / L, sucrose 10 to 30 g / L, agar 4 to 8 g / L Culturing in an included medium to form a plant; and

3) 0.1 to 3 mg / L of indole-3-butyric acid (INDOLE-3-BUTYRIC ACID, with or without addition of 1/10 of the zeatin riboside in the medium of step 2); Culturing in medium to which IBA) is added, or purifying in-flight in vitro to induce root formation.

The leaf culture technology of the present invention is cultured by using the leaves (leaflets) of the blueberry seedlings being cultured in the plane, so that it is easy to cultivate relatively simple, without the need for highly precise harvesting techniques such as growth point harvesting. In addition, since the formation rate of multi-shoot is higher than the growth point culture, it is advantageous to increase the number of seedlings in the growth culture process of expanding the culture scale. It can be used as a source technology.

1 is a diagram showing the leaf culture results of the blueberry Toro cultivar.
Figure 2 is a diagram showing the leaf culture results of blueberry legacy varieties.
Figure 3 is a diagram showing the leaf culture results of blueberry Oregon blue varieties.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for forming plants of blueberry varieties using the leaf culture method comprising the following steps.

1) Blueberry leaves (leaflets) 1 to 3 mg / L Zeatin, FeNa-EDTA 30 to 60 mg / L, Myo-Inositol 50 to 150 mg / L, sucrose Inducing initial callus and polycytheria by flocculation in a medium containing 10 to 30 g / L and 4 to 8 g / L of agar;

2) Zeatin Riboside 1 to 3 mg / L, FeNa-EDTA 30 to 60 mg / L, Myoinositol 50 to 150 mg / L, Thiamine HCl (Thiamine HCl) 1 in the medium of step 1) To 3 mg / L, nicotinic acid 0.5 to 1.5 mg / L, pyridoxine HCl 0.5 to 1.5 mg / L, sucrose 10 to 30 g / L, agar 4 to 8 g / L Culturing in an included medium to form a plant; and

3) 0.1 to 3 mg / L of indole-3-butyric acid (INDOLE-3-BUTYRIC ACID, with or without addition of 1/10 of the zeatin riboside in the medium of step 2); Incubating in medium to which IBA) is added, or purifying from in-flight (culture room) to in-flight (greenhouse) to induce root formation.

The blueberry variety may be, but not limited to, Toro, Legacy, and Oregon Blue.

The plant forming method may further include step 4) below, but is not limited thereto.

4) The step of culturing in the medium of step 3) to form a plant with roots from the cabin (culture room) to the outside (greenhouse).

When the blueberries are Toro or legacy varieties, FeNa-EDTA of step 1) may be 30 to 40 mg / L, but is not limited thereto.

When the blueberry is a Toro variety, FeNa-EDTA of step 2) may be 30 to 40 mg / L, but is not limited thereto.

If the blueberry is a legacy or Oregon blue variety, FeNa-EDTA of step 2) may be 50 to 60 mg / L, but is not limited thereto.

The medium of step 1) may be WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins), but is not limited thereto.

When the blueberry is an Oregon blue variety, the medium of step 1) is WPM medium or AN (Anderson Salt Medium) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free) It may be to use a medium in which the medium is mixed in a 1: 1 ratio, but is not limited thereto.

When the blueberry is a Toro variety, the medium of step 2) may be WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins), but is not limited thereto.

When the blueberry is a legacy or Oregon blue variety, the medium of step 2) is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins) and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt) Mixture, NH ₄ NO ₃ Free) medium may be used to mix the medium in a 1: 1 ratio, but is not limited thereto.

In a specific embodiment of the present invention, in order to form a seedling of the blueberry Toro cultivar, the leaves (leaflets) are Zeitan (Zeatin) 2 mg / L, FeNa-EDTA 36.7 mg / L, Myo-Inositol Initial callus and polycinus were induced by WPM medium supplemented with 100 mg / L, 20 g / L sucrose, and 6 g / L agar, and Zetin Riboside was added to the WPM medium. ) 2 mg / L, FeNa-EDTA 36.7 mg / L, Myoinositol 100 mg / L, Thiamine HCl 2 mg / L, Nicotinic acid 1 mg / L, Pyridoxine HCl (Pyridoxine HCl) 1 mg / L, sucrose 20 g / L, agar 6 g / L 8 species were cultured in the medium was added to form a seedlings, with or without addition of the concentration of the gelatin riboside 1/10, Indole-3-butyric acid (INDOLE-3-BUTYRIC ACID, IBA) was induced in roots in medium supplemented with 1-2 mg / L.

To form seedlings of blueberry legacy varieties, leaves (leaflets) were treated with gelatin 2 mg / L, FeNa-EDTA 36.7 mg / L, myoinositol 100 mg / L, sucrose 20 g / L, agar 6 g / L The initial callus and polycytheria were induced by the addition of WPM medium and the medium was mixed with WPM medium and MS medium in a 1: 1 ratio of 2 mg / L of zetin riboside, 36.7 mg / L of FeNa-EDTA, and myo Seedlings were formed by incubating in medium containing 100 mg / L of inositol, 2 mg / L thiamine HCl, 1 mg / L nicotinic acid, 1 mg / L pyridoxine HCl, 20 g / L sucrose, and 6 g / L agar. Roots were induced in medium with 1 to 2 mg / L of indole-3-butyric acid instead or with or without adding a concentration of 1/10 of the siatin riboside.

To form a seedling of the Blueberry Oregon Blue variety, the leaves (leaflets) were treated with gelatin 2 mg / L, FeNa-EDTA 36.7 mg / L, myoinositol 100 mg / L, sucrose 20 g / L, agar 6 g / WPM medium with 4 L or gelatin 2 mg / L, FeNa-EDTA 36.7 mg / L, myoinositol 100 mg / L, sucrose 20 g / L, agar 6 g / L and AN medium and MS medium Initial callus and polycythemia were induced by instillation on a medium mixed in a 1: 1 ratio, and 2 mg / L of zetin riboside and 36.7 mg / FeNa-EDTA in a medium mixed with a WPM medium and an MS medium in a 1: 1 ratio. L, myoinositol 100 mg / L, thiamine HCl 2 mg / L, nicotinic acid 1 mg / L, pyridoxine HCl 1 mg / L, sucrose 20 g / L, agar 6 g / L Roots were induced in the medium to which 1 to 2 mg / L of indole-3-butyric acid was added, with or without adding the concentration of zitin riboside to 1/10.

Therefore, two highbush varieties (Toro, Legacy) and one rabbit eye type (Oregon Blue), which are highly adaptable and occupy a relatively large growing area, are leaf-cultured to form seedlings within 8 to 9 months. And the development of culture conditions, it can be used as a source technology for the expansion and spread of blueberry domestic tissue culture seedlings.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Toro Varieties Leaf culture  Way

<1-1> Early Toro Varieties Callus ( clallus A) forming and Rechincho ( mutli - shoot ) Induction stage

The leaves of seedlings of the Blueberry Toro cultivar in inflight were cut to remove leaf stalks, and then leveled so that the upper surface of the leaves reached the initial callus forming medium. The initial callus and polycythemia induction medium was prepared by adding four additives, such as growth regulator Zeatin, to WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins) (MBcell) (Table 1 ). Each medium component was prepared by mixing, dispensing 80 mL each in a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized for 15 minutes at 121 ℃. Fifteen leaves were healed in a culture bottle containing medium, and after culturing for 2 weeks under dark conditions in a culture room maintained at 25 ± 1 ° C., 14 ~ 16 weeks under light conditions (2,000 ~ 3,000 lux, 16 hours length) Incubated.

As a result, it was confirmed that callus and polycytheria were induced from the leaves of the blueberry Toro varieties densely wound on the WPM medium.

Name content WPM Badge 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 36.70 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 Sucrose 20 g / L Zeatin 2.00 mg / L Agar 6.0 g / L The pH of the medium 5.0

basic
Badge Growth
Moderator Callus formation rate (%) Differentiation rate of shoots (%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM Giatin 2.00mg / L 66.3 ± 5.8 6.6 ± 0.8 3.5 ± 0.6 20.4 ± 0.4

<1-2> Plant Formation Steps of Toro Varieties

16 ~ 18 weeks after leaf healing, after initial callus formation and polycystic induction culture, plantlets are formed and plant regeneration when cultured for 4 ~ 6 weeks after passage to the culture bottle containing the medium of <Table 3>. Induced.

As a result, when cultured in the medium of Table 3, it was confirmed that 4 ~ 6 weeks after the seedling formation and plant regeneration was induced, the characteristics of the seedlings formed at 24 weeks are as shown in Table 4 below.

Name content WPM Badge 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 36.70 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 Myoinositol 100 mg / L Nicotinic acid 1.0 mg / L Pyridoxine HCl 1.0 mg / L Thiamine HCl 2.0 mg / L Sucrose 20 g / L Zeatin Riboside 2.00 mg / L Agar 6.0 g / L The pH of the medium 5.0

basic
Badge Growth
Moderator Plant formation rate
(%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM Giatin riboside2.00mg / L 65.0 ± 7.1 3.6 ± 0.8 36.7 ± 3.5

<1-3> Root Formation of Toro Varieties ( Rooting ) step

In order to form seedling roots of Toro cultivar, the concentration of Zein Ribodise was reduced to 1/10 in the medium composition of <Table 3> or IBA 1 or 2 mg / L was added to the medium without addition. It was used as a rooting medium, and cultured for about 1 month. In this case, when the culture bottle stopper with a cotton blocking hole is used to circulate the outside air can increase the survival rate during the acclimatization. At this time, when the total length of the seedlings was sufficiently grown to 5 cm or more, it was possible to directly purified to the outside (greenhouse) without going through the rooting culture to form the roots.

Rooted seedlings or roots were not formed through rooting culture, but Toro seedlings grown more than 5 ㎝ did not form the complete tissue culture seedlings after the process of cultivation from in-flight (culture room) to outside (greenhouse). Prepare top soil mixed with peat moss and pearlite 1: 1 (v: v), and then plant blueberry seedlings in pots (internal diameter 110 mm, height 113 mm), and shade 30-40% of sunlight, up to 25 ℃ In the greenhouse maintained at a minimum of 18 ℃ covered the transparent vinyl was cured in a condition that keeps the humidity well. While checking the condition of the seedlings, the vinyl was slowly rolled out and exposed to the outside air to completely cure.

As a result, when the concentration of Zeatin Ribodise was reduced to 1/10 in the medium composition of <Table 3> or IBA 1 or 2 mg / L was added to the medium without addition, it was used as the rooting medium. It was confirmed that the roots of the Toro varieties were formed, and when subjected to the step of hardening to the outside, it was confirmed that the adaptation to the outside environment (Fig. 1).

< Example  2> Legacy  Varietal Leaf culture  Way

<2-1> Legacy  Early years of breed Callus  Formation and Rechincho  Induction stage

The seedling leaves of the blueberry legacy varieties in-flight were cut to remove leaf stalks and horizontally so that the top surface of the leaves reached the initial callus forming medium. The initial callus and polycythemia induction medium was prepared by adding growth regulator giatin and four additives to the WPM medium. The medium composition component is the same as said <Table 1>. Each medium component was prepared by mixing, dispensing 80 mL each in a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized for 15 minutes at 121 ℃. Fifteen leaves were healed in a culture bottle containing medium, and after culturing for 2 weeks under dark conditions in a culture room maintained at 25 ± 1 ° C., 14 ~ 16 weeks under light conditions (2,000 ~ 3,000 lux, 16 hours length) Incubated.

As a result, it was confirmed that callus and polycytheria were induced from the leaves of the blueberry legacy varieties dented on the WPM medium, and the callus formation and shoot differentiation characteristics at the 18th week are shown in Table 5 below.

basic
Badge Growth
Moderator Callus formation rate (%) Differentiation rate of shoots (%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM Giatin 2.00mg / L 49.7 ± 4.4 10.4 ± 2.3 11.3 ± 2.2 21.7 ± 4.5

<2-2> Legacy  Plant formation stages of varieties

16 ~ 18 weeks after leaf tooth formation, after initial callus formation and polycystic induction culture, the seedlings and plant regeneration were induced for 4 ~ 6 weeks by passage to the culture bottle containing the medium of <Table 3>.

As a result, when cultured in the medium of <Table 6>, it was confirmed that 4 ~ 6 weeks after the seedling formation and plant regeneration was induced, the characteristics of the seedlings formed at 24 weeks are shown in Table 7 below.

Name content WPM: MS (1: 1) 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 Myoinositol 100 mg / L Nicotinic acid 1.00 mg / L Pyridoxine HCl 1.00 mg / L Thiamine HCl 2.00 mg / L Sucrose 20 g / L Giatin riboside 2.00 mg / L Agar 6.0 g / L The pH of the medium 5.0

basic
Badge Growth
Moderator Plant formation rate
(%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM: MS
(1: 1) Giatin riboside 2.00 mg / L 46.0 ± 3.3 13.6 ± 2.7 37.7 ± 2.1

<2-3> Legacy  Root formation of varieties ( Rooting ) step

In order to form seedling roots of legacy varieties, the concentration of zitin riboside (Zeatin Ribodise) was reduced to 1/10 or the addition of IBA 1 or 2 mg / L It was 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. At this time, when the total length of the seedlings was sufficiently grown to 5 cm or more, it was possible to directly purified to the outside (greenhouse) without going through the rooting culture to form the roots.

Rooted seedlings or roots were not formed through rooting cultivation, but legacy seedlings grown more than 5 ㎝ did not form the complete tissue culture seedlings after undergoing the process of in-flight incubation. Prepare top soil mixed with peat moss and pearlite 1: 1 (v: v), and then plant blueberry seedlings in pots (internal diameter 110 mm, height 113 mm), and shade 30-40% of sunlight, up to 25 ℃ In the greenhouse maintained at a minimum of 18 ℃ covered the transparent vinyl was cured in a condition that keeps the humidity well. While checking the condition of the seedlings, the vinyl was slowly rolled out and exposed to the outside air to completely cure.

As a result, when the concentration of zitin riboside (Zeatin Ribodise) was reduced to 1/10 in the medium composition of <Table 3> or IBA 1 or 2 mg / L was added to the medium without addition, it was used as the rooting medium. It was confirmed that the roots of the legacy varieties are formed, and when subjected to the step of curing in vitro, it was confirmed that the adaptation to the outside environment (Fig. 2).

< Example  3> Oregon Blue  Varietal Leaf culture  Way

<3-1> Oregon Blue  Early years of breed Callus  Formation and Rechincho  Induction stage

The leaves of the seedlings of the blueberry Oregon blue cultivar in the cabin were cut to remove the petioles, and then horizontally soaked so that the upper surface of the leaves could reach the initial callus forming medium. Initial callus and polycythemia induction medium was WPM medium or Anderson Salt Medium (AN) (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 additives Giatin (Zeatin) and four additives to the medium mixed in a 1: 1 ratio. The composition of the medium is as shown in Table 8 above.

Name content WPM Badge AN: MS (1: 1) badge NH ₄ NO ₃ 300.00 mg / L 200.00 mg / L MgSO ₄ 180.60 mg / L 180.62 mg / L KNO ₃ 475.00 mg / L 1190.00 mg / L K ₂ SO ₄ 742.50 mg / L 0.00 mg / L KH ₂ PO ₄ 170.00 mg / L 85.00 mg / L _{Ca (NO 3) 2 4H 2} O 289.50 mg / L 0.00 mg / L CaCl ₂ 137.38 mg / L 332.11 mg / L CoCl ₂ 6H ₂ O 0.00625 mg / L 0.025 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 16.90 mg / L H ₃ BO ₃ 6.20 mg / L 6.20 mg / L KI 0.2075 mg / L 0.565 mg / L FeNaEDTA 36.70 mg / L 55.05 mg / L Na ₂ EDTA 27.97 mg / L 37.25 mg / L FeSO ₄ 7H ₂ O 20.88 mg / L 27.85 mg / L CuSO ₄ 5H ₂ O 0.19 mg / L 0.025 mg / L Myoinositol 100.00 mg / L 100.00 mg / L Sucrose 20.00 g / L 20.00 g / L Giatin 2.00 mg / L 2.00 mg / L Agar 6.00 g / L 6.00 g / L The pH of the medium 5.0 5.0

Each medium component was prepared by mixing, dispensing 80 mL each in a culture bottle (outer diameter 81 mm, height 132 mm) and sterilized for 15 minutes at 121 ℃. Fifteen leaves were healed in a culture bottle containing medium, and after culturing for 2 weeks under dark conditions in a culture room maintained at 25 ± 1 ° C., 14 ~ 16 weeks under light conditions (2,000 ~ 3,000 lux, 16 hours length) Incubated.

As a result, it was confirmed that callus and polycytheria were induced from the leaves of the blueberry Oregon blue varieties densified in the <Table 8> medium, and the initial callus formation and shoot differentiation characteristics of the 18th week were as shown in Table 9 below.

basic
Badge Growth
Moderator Callus formation rate
(%) Shoot differentiation rate
(%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM Giatin 2.00mg / L 71.4 ± 10.6 11.6 ± 3.8 14.4 ± 2.4 13.1 ± 4.9 AN: MS (1: 1) Giatin 2.00mg / L 56.9 ± 6.9 11.5 ± 2.6 17.1 ± 0.2 19.9 ± 5.1

<3-2> Oregon Blue  Plant formation stages of varieties

16 ~ 18 weeks after leaf formation, after initial callus formation and polycythemia-induced culture, plantlets were formed for 4 ~ 6 weeks after passage to the culture bottle containing the medium of <Table 6> And plant regeneration.

As a result, when cultured in the medium of <Table 6>, it was confirmed that 4 ~ 6 weeks after the seedling formation and plant regeneration was induced, the characteristics of the seedlings formed at 24 weeks are shown in Table 10 below.

basic
Badge Growth
Moderator Plant formation rate
(%) Fresh water
(Dog / plantlet) New plant height
(mm) WPM: MS
(1: 1) Giatin riboside 2.00 mg / L 68.4 ± 3.4 16.9 ± 1.0 39.6 ± 5.9

<3-3> Oregon Blue  Root formation of varieties ( Rooting ) step

To form seedling roots of Oregon Blue varieties, IBA 1 or 2 mg / L was added to the medium without reducing the concentration of Zetin Ribodise to 1/10 in the medium composition of <Table 3>. Was 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. At this time, when the total length of the seedlings sufficiently grown to 5 cm or more, it was possible to immediately purified to the outside without going through the rooting culture to form the roots.

Rooted seedlings or roots were not formed through rooting cultivation, but Oregon blue seedlings grown more than 5 cm did not have the form of complete tissue culture seedlings after the in-flight purifying process. Prepare top soil mixed with peat moss and pearlite 1: 1 (v: v), and then plant blueberry seedlings in pots (internal diameter 110 mm, height 113 mm), and shade 30-40% of sunlight, up to 25 ℃ In the greenhouse maintained at a minimum of 18 ℃ covered the transparent vinyl was cured in a condition that keeps the humidity well. While checking the condition of the seedlings, the vinyl was slowly rolled out and exposed to the outside air to completely cure.

As a result, when the concentration of zitin riboside (Zeatin Ribodise) in the medium composition of Table 6 was reduced to 1/10 or IBA 1 or 2 mg / L was added to the medium without addition, it was used as the rooting medium. It was confirmed that the roots of Oregon Blue varieties are formed, and when subjected to the step of curing outside, it was confirmed that the adaptation to the outside environment (Fig. 3).

Claims (7)

Method of plant formation of blueberry varieties using leaflet culture method comprising the following steps:
1) Blueberry leaves (leaflets) 1 to 3 mg / L Zeatin, FeNa-EDTA 30 to 60 mg / L, Myo-Inositol 50 to 150 mg / L, sucrose Inducing initial callus and polycytheria by flocculation in a medium containing 10 to 30 g / L and 4 to 8 g / L of agar;
2) Zeatin Riboside 1 to 3 mg / L, FeNa-EDTA 30 to 60 mg / L, Myoinositol 50 to 150 mg / L, Thiamine HCl (Thiamine HCl) 1 in the medium of step 1) To 3 mg / L, nicotinic acid 0.5 to 1.5 mg / L, pyridoxine HCl 0.5 to 1.5 mg / L, sucrose 10 to 30 g / L, agar 4 to 8 g / L Culturing in an included medium to form a plant; and
3) 0.1 to 3 mg / L of indole-3-butyric acid (INDOLE-3-BUTYRIC ACID, with or without addition of 1/10 of the zeatin riboside in the medium of step 2); Incubating in medium to which IBA) is added, or purifying from in-flight (culture room) to in-flight (greenhouse) to induce root formation.
The method of claim 1, wherein the blueberry varieties are Toro, Legacy, and Oregon Blue.
The method of forming a plant according to claim 1, further comprising the following step 4):
4) The step of culturing in the medium of step 3) to form a plant with roots from the cabin (culture room) to the outside (greenhouse).
The method of claim 1, wherein the medium of step 1) is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins).
According to claim 1, wherein the blueberry is Oregon blue variety, the medium of step 1) WPM medium or AN (Anderson Salt Medium) medium and MS (Murashige & Skoog Medium, Mod.No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free) plant formation method characterized in that the medium is mixed with a 1: 1 ratio.
The method of claim 1, wherein the medium of step 2) is WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins).
According to claim 1, wherein the blueberry is a Legacy or Oregon Blue variety, the medium of step 2) WPM medium (Lloyd & McCown Woody Plant Basal Salt Medium w / o vitamins) and MS (Murashige & Skoog Medium, Mod) No. 4 Basal Salt Mixture, NH ₄ NO ₃ Free) Plant forming method characterized by using a medium in which the medium is mixed in a 1: 1 ratio.

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