KR20170095705A - Mass production method of Black currant nursery tree by in vitro culture - Google Patents

Abstract

The present invention relates to a method for mass-producing Ribes nigrum seedlings by using an in-vitro culture scheme. According to the present invention, the method includes the steps of: (a) obtaining and sterilizing the young buds of the Ribes nigrum seedlings; (b) forming new sprouts by culturing the sterilized young buds in a new sprout culture medium; (c) culturing and growing the new sprouts for 28 to 35 days after moving the new sprouts cut in node units to a seedlings sprout medium to obtain node seedlings; (d) culturing the node seedlings in a node seedling culture medium to increase the height and diameter thereof; and (e) culturing and growing the grown node seedlings in a root growing medium. The present invention can enable mass production of the Ribes nigrum seedlings by using the in-vitro culture scheme on the young buds thereof and grow the mass-produced Ribes nigrum in soil to grow the Ribes nigrum seedlings.

Description

[0001] The present invention relates to a method for mass production of black currant seedlings by in-

The present invention relates to a method for mass production of black currant seedlings by in-vitro culturing in which culturing conditions for in-flight culture are established to mass-produce black currant.

Black currant is a deciduous broad-leaved shrub that originates in central northern Europe and the Himalayas, and grows to a height of 1 to 2 meters. The leaves of black currant are 3 ~ 5㎝ in length and width, are divided into 3 ~ 5 pieces, have irregular mounts, have no hairs on the leaf surface, scatter on the back side, and have incense on leaves and stem. The flowers of black currant are 4 ~ 6mm in diameter, reddish brown, and 5 ~ 10cm in length. The fruit of blackcurrant is ripened in July ~ August, diameter is about 1cm, the surface is shiny, and when it is ripe, it has a dark blue color close to black.

Black currants have been used as edible and nutritious food for centuries. In northern Europe, black currants have been consumed as edible fruits from ancient times. Medicinal herbs have been used since the 16th century, and herbalists have been using Blackcurrant to treat bladder stones and have been using blackcurrant as a syrup for cold and respiratory diseases.

Blackcurrant began to spread throughout the world in the 18th century. Especially in Europe, it has been widely used among herbalists and doctors. It has been used for the treatment of various diseases such as intestinal treatment, typhoid, rheumatism, arthritis, mouth treatment and skin urinary treatment.

In the 20th century, the black currant was dried and drunk with tea. Drinking tea has the effect of stopping the plague and preventing bleeding from vitamin C deficiency. It has also been used for asthma, diarrhea, inflammation of the gums, diuretic and nutritional tonic.

The health effects of blackcurrant were scarcely known scientifically until the late 1970s, and since then many studies have shown that they contain important chemical components needed to enhance the immune system of our body. Based on the ingredients contained in Blackcurrant, the development of therapeutic agents using Blackcurrant is proceeding.

Black currants contain the largest amount of vitamin C in the world's fruit, four times more than the orange known as vitamin C. In addition, black currants contain various bioflavonoids, and it is known that the content of anthocyanins is significantly higher than that of other fruits. Large amounts of anthocyanins have the effect of improving vision and reducing the aging-related illnesses.

Based on the ingredients of Blackcurrant, the development of a therapeutic agent using blackcurrant is proceeding.

Recent studies have shown that blackcurrant extracts have an antioxidant effect, and the high sulfation (anti-aging) efficacy of blackcurrants, anti-aging effects, (Free radicals) and neutralize the active oxygen and prevent the damage of cells and DNA has been found. It has also been demonstrated that platelet aggregation is inhibited, that it has some loosening effect on smooth muscle, and that it reduces the factors for chronic inflammatory diseases.

Blackcurrant has been shown to be effective in arteriosclerosis, arthritis, rheumatism, prostate cancer, menopause, and throat pain. It is known to inhibit inflammation such as atherosclerosis, emphysema, rheumatoid arthritis and the like by inhibiting the chemical reaction by enzymes such as elastase which can cause collagen degradation. Studies on inhibition and treatment of cancer by using black currant have been conducted, and the effect on cancer has already been proven in many cases. It has also been reported that an anti-influenza effect against influenza A virus has been reported. In addition, research is being conducted as a therapeutic agent for Alzheimer's disease, heart disease, microbial infection and neurological diseases.

Among the various blackcurrant varieties in Korea, ebony cultivars are the most cultivated. Ebony varieties are known to contain a large amount of vitamin C (302% of daily intake per 100g) compared to other fruits and are reported as potassium, phosphorus, iron, vitamin B5 and a wide range of essential nutrients.

The blackcurrant fruit of sour taste is mainly used as a raw material for reproductive use, jam, juice, ice cream and so on, and is used as various food additives. In the UK, it is mixed with cider or mixed with beer. Ribena juice is famous for black currant drink. In Russia, leaves are also used by car. In addition, blackcurrant seed oil is rich in nutrients, especially unsaturated fatty acids such as alpha-linolenic acid and gamma linolenic acid and vitamin E. Studies have also shown that breastfeeding mothers can reduce atopic dermatitis in children by consuming blackcurrant seed oil.

In Korea, black currant is imported and cultivated as an abalone seedlings. In the case of abalone seedlings, life span is short, which leads to weakness of water quality and deterioration of quality.

Korean Patent Publication No. 10-2011-0080998

In order to solve the above problems, it is an object of the present invention to provide a method of mass-producing black currant seedlings by cultivating a large amount of black currant young eyes in the cabin and transplanting them in soil.

In order to achieve the above object, according to the present invention,

(a) collecting and sterilizing young eyes of a black current seedlings;

(b) 1 liter of MS (murashigae and Skoog) basal medium Culturing the sterilized young eyes in a shoot formation medium supplemented with 20 to 50 g of sucrose and 2 to 5 g of phytagel to form shoots;

(c) The nodules prepared by cutting the formed shoots into nodules were suspended in 1 L of MS basal medium Transferring it to Seedling seedlings supplemented with 1 ~ 5 mg of BAP and culturing for 28 ~ 35 days to obtain seedlings.

(d) seeding the seedlings at a rate of 1 < RTI ID = 0.0 > Culturing the seedlings in a seedling growth medium supplemented with 0.1 to 3 mg of BAP to grow key and diameter; And

(e) culturing the grown seedling seedlings at a concentration of 1 < RTI ID = 0.0 > Culturing in a rooting medium supplemented with 1 to 3 mg of one selected from among indole butyric acid (IBA), 1-naphthalene acetic acid (NAA) and indole acetic acid (IAA) Mass production method.

The method may further comprise the step of purifying the rooted seedlings obtained in the rooting step to the soil.

In this method, the disinfection may be carried out by a method comprising primary sterilization with a surfactant, washing with distilled water, secondary disinfection with alcohol, final disinfection with sodium hypochlorite and washing with distilled water .

In this method, for the shoot formation, it is preferable that the young eyes are cultured in the shoot formation medium at 22-27 째 C and 2000-3000 lux for 15-17 hours with light irradiation for 14-21 days.

In the above method, it is preferable to cultivate the nodule shoots at 22 to 27 ° C in seedling seedlings at 2000 to 3000 lux for 15 to 17 hours while irradiating the shoots for 28 to 35 days.

In the above method, in order to grow the seedling, it is preferable that the seedling is cultured in a seedling growth medium at 22 to 27 DEG C for 15 to 17 hours at 2000 to 3000 lux for 14 to 21 days.

In this method, for the rooting, it is preferable to cultivate the seedling seedlings in a rooting medium at 22-27 ° C at 2000-3000 lux for 15-17 hours with light irradiation for 13-15 days.

According to the method of the present invention, black currants can be mass-grown by in-vitro culturing of black currant young eyes, and black currants can be grown in large quantities by transplanting large-scale black currants into soil. Therefore, it is possible to solve problems such as short life span, weak wash, low quality, and the like when the cutting is imported and cultivated.

FIG. 1 is a photograph showing a state in which contamination is removed by disinfection after collecting black currant young eyes. FIG.
Fig. 2 is a photograph showing a state in which shoots are formed in young eyes of black currant. Fig.
3 is a photograph showing a state in which black currant seedlings were grown.
Fig. 4 is a photograph showing the state of the seedlings in which the propagated black current shoots were rooted.
FIG. 5 is a photograph showing a state in which rooted black currant seedlings are purified in soil.

The method for mass production of black currant seedlings by in-vitro culture of the present invention will be described in detail as follows.

(1) Collection and disinfection of young eyes

To mass-produce black currants, first collect young eyes from the packaging. The branches of the youngest part of the black current tree are taken from branches that have not undergone lignification, and the leaves are removed from the harvested branches to collect young eyes.

Disinfect the young eyes and remove the foreign contaminants from the young eyes. A preferred disinfection method is as follows.

First, the eyes of young branches taken from the package are trimmed and sterilized using a surfactant. The surfactant is preferably used in the form of a liquid mixed with sterilized water. An example of the surfactant is Triton X-100, and it is more preferable to use the surfactant in the form of a solution containing 1 to 2 drops of Triton X-100 in sterilized water. The primary disinfection is preferably repeated 2-3 times. After first disinfection, wash with distilled water.

After washing with distilled water, secondly sterilize with alcohol for 40 seconds to 2 minutes. It is preferable to use 70% alcohol, and it is more preferable to sterilize with 70% alcohol for 1 minute.

After the second disinfection, final disinfection is carried out using sodium hypochlorite. It is preferable to use 0.5 to 2% solution of sodium hypochlorite. After the final disinfection, the disinfectant is completely removed by washing with sterilized water 3 to 5 times

By the disinfection process as described above, it is possible to improve the efficiency of mass proliferation and to perform healthy cultivation by removing foreign contaminants in the young eyes of the black current branches.

(2) Shoot  Forming step

The sterile young eyes are cultured in a shoot-forming medium. As a shoot-forming medium, the medium (Murashige and Skoog) A medium supplemented with sucrose and Phyta gel is used.

The sucrose grows young eyes and acts as an energy source to form shoots. When sucrose is used in an amount of less than 20 g, the germinated shoots of the seeds do not grow normally due to insufficient energy sources required for normal growth, and when sucrose is used in an amount exceeding 50 g A problem arises in that the energy source necessary for normally growing the germinated shoots is excessively large.

The phytagel serves to gellify (solidify) the liquid MS base medium to allow the seeds to germinate by being fixed in place without stagnation. It is preferable to add 2-5 g of phytagel per 1 L of MS base medium. If less than 2 g of phytagel is used, the MS base medium in the liquid phase will not gel, and if it is used in excess of 5 g, So that the seed is germinated.

The sterilized young eyes were cultured in a shoot-forming medium at 22-27 ° C for 12-23 days with light irradiated at 2000-3000 lux for 15-17 hours to form shoots.

(3) Shoot  Propagation step

When shoots are formed as described above, shoots are cut in each node unit, and the cut nodules are transferred to seedling seedlings and grown to obtain node seedlings.

As a new seed culture medium for growing shoots, a medium supplemented with BAP in MS base medium is used.

BAP (benzylaminopurine) plays a role as a growth regulator mainly for the growth of shoots. BAP is preferably added in an amount of 1 to 5 mg per liter of MS basic medium. If less than 1 mg is used or more than 5 mg is used, shoot formation and propagation of seedlings can not be normally performed.

The shoots cut at each nodule unit are transferred to the seedling seedling supernatant and cultured at 22 to 27 ° C for 15 to 17 hours at 2000 to 3000 lux for 28 to 35 days for proliferation.

(4) Mardi  Growth stage

The shoots of the shoots are transferred to the seedling growth medium to grow the height and diameter of the seedlings.

As the medium for seedling growth, a medium supplemented with BAP in MS base medium is used.

BAP plays an important role in the growth of seedlings as well as the growth of seedlings. BAP is used by adding 0.1 ~ 3mg per 1L of MS base medium. When using less than 0.1mg, the height and diameter of seedlings grow too slow and when the seeds are used in excess of 3mg, This is because they are not healthy seedlings.

The seedlings were cultured in the seedling seedling growth medium at 22-27 ° C at 2000-3000 lux for 15-17 hours for 14-21 days to grow the seedlings.

(5) Mardi Stage of rooting

When the seedling grows, the seedlings are transferred to the rooting medium to induce rooting. As the rooting medium, a medium supplemented with a selected one of IBA, NAA and IAA (indole acetic acid) is used as a MS basic medium.

The additives IBA, NAA and IAA both play a role in promoting and growing the rooting of the node seedlings. IBA, NAA and IAA are preferably added in an amount of 1 to 3 mg per 1 liter of MS base medium. When the amount of IBA, NAA and IAA is less than 1 mg, rooting of the seedlings becomes too slow. When the seeds are used in excess of 3 mg, It is because it can not be rooted.

The seedlings were cultured in the rooting medium at 22 to 27 ° C. for 13 to 15 days under light irradiation at 2000 to 3000 lux for 15 to 17 hours.

(6) Soil purification stage

The black currant seedlings produced as described above can be cultivated in soil in a conventional manner.

Peat moss: vermiculite: Perlite is blended at a weight ratio of 3: 1: 1, sterilized and placed in a pest box. When three or more fine roots occur at the rooting stage of the node seedlings, the medium adhered to the roots is removed, the seedlings from which the medium is removed are transferred to the prepared purifying box, and after one week, the lid of the purifying box It is slightly opened to expose to the growth environment of the plant growth chamber or the growth chamber. The reason why the above soil, vermiculite and pearlite are blended in a weight ratio of 3: 1: 1 is that the soil, vermiculite, and pearlite serve to promote smooth rooting and growth of the root through moisture supply, air permeability and nutrient supply of seedling In order to adapt the plant grown in the cabin to the external environment, moisture content, ventilation and nutrition should be appropriately supplied. In particular, vermiculite and pearlite are used for imparting water content and air permeability to soil, and soil is used for nutrition supply of seedlings. If the above mixing ratio is not applied, the growth of seedlings is delayed, And pearlite is preferably in a weight ratio of 3: 1: 1

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

< Example  1>

Collection and sterilization of young eyes

For mass production of black currant, young eyes were first taken from the packaging. The youngest branches of blackcurrant trees were harvested and the leaves were removed from the harvested branches. The leaf-removed branches were sterilized by sterilization three times using sterile water containing 1 ~ 2 drops of surfactant Triton X-100, washed with distilled water, sterilized with 70% alcohol for 1 minute, Sodium chlorate 0.5%, 1.5%, and 2.0% solution was used one time in order, and after the final disinfection, it was washed 4 times with sterilized water to completely remove the disinfectant solution. A photograph of a black currant young eye in which contamination is removed by disinfection after collection is shown in FIG.

< Experimental Example  1>

Check the effect of disinfectant

The effect of disinfection according to the concentration of sodium hypochlorite solution and addition of surfactant upon disinfection of the young eyes was confirmed as follows.

The disinfection effect of sodium hypochlorite solution was compared between 0.5%, 1.5%, and 2.0% sodium hypochlorite solution and 0.5 ~ 2% concentration . Triton X-100 was used as a surfactant and the effects of addition of 1 to 2 drops of Triton X-100 in sterilized water were compared. The results are shown in Table 1 below.

division Contamination rate (%) 0.5% solo 1.5% stand alone 2.0% exclusive 0.5 to 2.0% mixed No added Triton X-100 90 ± 0.5 88 ± 0.6 75 ± 0.5 50 ± 0.3 Addition of Triton X-100 70 ± 1.0 55 ± 1.0 32 ± 1.2 5 ± 0.2

As shown in Table 1, mixing of the sodium hypochlorite solution with the concentration of sodium hypochlorite solution at a concentration lower than that of the sodium hypochlorite solution alone decreased the contamination rate of black currant, and addition of Triton X-100 did not add The contamination rate of black currant was significantly reduced compared with the case without treatment.

< Example  2>

Shoot  formation

Aseptic young eyes obtained by disinfection as described above were cultured in a shoot-forming medium.

As the shoot-forming medium, 35 g of sucrose and 3.5 g of phytase gel were added per liter of MS base medium. The sterilized young eyes were cultured in shoot shoot formation medium at 25 ° C for 16 days with light irradiation at 2500 lux for 18 days to form shoots. A photograph of the shoots formed is shown in Fig.

< Example  3>

Shoot  multiplication

The shoots were cut at each node unit, and the cut nodules were transferred to seedling seedlings and seedlings were propagated to obtain seedlings.

As a new seed culture medium for growing shoots, a medium supplemented with 3 mg of BAP per 1 liter of MS base medium was used. The seedlings were transferred to the seedling seedlings at 25 ° C for 16 hours at 25 ° C for 35 days.

< Experimental Example  2>

Seedling  Following Shoot  Confirmation of proliferation effect

In order to investigate the growth effect of shoots according to the components added to Seedling seedlings, the following experiment was conducted.

(2) medium 2 (medium supplemented with 3 mg of BAP and 0.3 mg / l of MS basic medium, and (3) medium supplemented with 3 mg of BAP per 1 l of MS basic medium); and (3) The shoots were grown using Medium 3 (medium supplemented with 3 mg of BAP and 0.3 mg of NAA per liter of MS base medium). 20 days, 30 days and 35 days, respectively. The results are shown in Table 2 below.

division Badge 1 Badge 2 Badge 3 Shoot growth Day of multiplication 20 3.5 ± 0.5cm 1 ± 0.5 cm 1 ± 0.5 cm 30 4.2 ± 1.2 cm 2.5 ± 1.3 cm 1.8 ± 1.2 cm 35 4.5 ± 1.5 cm 2.5 ± 1.3 cm 2.2 ± 0.6 cm

As shown in Table 2, the medium containing BAP (medium 1) was the most excellent for MS seed medium, and medium (medium 2) supplemented with BAP + IAA and BAP + NAA The added medium (medium 3) was similar. MS supplemented with BAP for 30-35 days was most preferable.

< Example  4>

Mardi  growth

The larvae of the shoots were transferred to the seedling growth medium and grown to the height and diameter.

As the seedling growth medium, a medium supplemented with 1 mg of BAP per liter of MS base medium was used. The proliferated seedlings were cultured in a seedling growth medium at 25 ° C for 16 days with light irradiation at 2500 lux for 18 days to form shoots. Fig. 3 shows a photograph of the seedling seedlings grown on the seedling seedling growth medium.

< Experimental Example  3>

On the seedling growth medium  Following Seedling  Confirmation of growth effect

The growth of the seedling was evaluated according to the amount of BAP added in the seedling growth medium as follows.

BAP was added at 0.1 mg, 1 mg, 2 mg and 3 mg per liter of MS basal medium, and the growth of the seedling was observed at 1 week, 3 weeks and 5 weeks. The results are shown in Table 3 below.

division BAP (mg / l) 0.1 One 2 3 1 week 0.53 + - 0.2 cm 0.54 ± 0.3 cm 0.48 ± 0.6 cm 0.52 ± 0.3 cm 3 weeks 0.95 + - 0.2 cm 1.97 ± 0.4 cm 0.85 ± 0.4 cm 0.78 ± 0.4 cm 5 weeks 1.33 ± 0.3 cm 2.32 ± 0.3 cm 1.22 ± 0.2 cm 1.02 + - 0.5 cm

As can be seen from the results in Table 3, when 1 mg of BPA was added, the growth of the seedling was the best.

< Example  5>

Mardi Rooting

The root and root diameter of the seedling were transferred to rooting medium to induce rooting.

As the rooting medium, a medium supplemented with 3 mg of IBA per liter of MS base medium was used. Mardi seedlings were cultured for 14 days at 25 ° C in a rooting medium at 2500 lux for 16 hours. Fig. 4 shows the state of the seedling seedlings rooted in the rooting medium.

< Experimental Example  4>

On rooting badge  Following Root effect  Confirm

The effects of IBA, NAA, and IAA, which are added to MS basal medium in rooting medium, on roots were confirmed as follows.

IBA, NAA and IAA were added at 1 mg, 2 mg and 3 mg, respectively, per liter of MS basal medium, and rooting amount was confirmed after 2 and 4 weeks. The results are shown in Table 4 below.

division MS basal medium + IBA (mg / l) MS basal medium + NAA (mg / l) MS basal medium + IAA (mg / l) One 2 3 One 2 3 One 2 3 2 weeks 0.3cm 0.5cm 1.8cm 0.3cm 0.4cm 0.8cm 0.1cm 0.2cm 0.5cm 4 weeks 1.8cm 1.2cm 2.8 cm 0.8cm 0.8cm 1.0cm 0.5cm 0.8cm 1.0cm

As can be seen from the results in Table 4 above, when IBA was added to MS base medium, rooting effect was better than when NAA or IAA was added. The addition of 3 mg of IBA per 1 L of MS basal medium resulted in the best rooting effect

< Example  6>

Purification in soil

The resulting black currant seedlings were cultivated in soil.

First, a purifying box is prepared by mixing peat moss: vermiculite: perlite in a weight ratio of 3: 1: 1, sterilizing and putting in a purifying box. When the number of fine roots is more than 3 to 5 in the step of rooting of the seedling, the seedling adhered to the roots is removed, and the seedlings with the medium removed are transferred to the prepared purifying box and after 1 week, the lid of the purifying box It is slightly opened and exposed to the external environment in a growth chamber or a growth chamber to purify the soil. A photograph of the purified black currant seedlings in soil is shown in Fig.

Claims (7)

(a) collecting and sterilizing young eyes of a black current seedlings;
(b) 1 liter of MS (murashigae and Skoog) basal medium Culturing the sterilized young eyes in a shoot formation medium supplemented with 20 to 50 g of sucrose and 2 to 5 g of phytagel to form shoots;
(c) The nodules prepared by cutting the formed shoots into nodules were suspended in 1 L of MS basal medium Transferring it to Seedling seedlings supplemented with 1 ~ 5 mg of BAP and culturing for 28 ~ 35 days to obtain seedlings.
(d) seeding the seedlings at a rate of 1 &lt; RTI ID = 0.0 &gt; Culturing the seedlings in a seedling growth medium supplemented with 0.1 to 3 mg of BAP to grow key and diameter; And
(e) culturing the grown seedling seedlings at a concentration of 1 &lt; RTI ID = 0.0 &gt; Culturing in a rooting medium supplemented with 1 to 3 mg of one selected from among indole butyric acid (IBA), 1-naphthalene acetic acid (NAA) and indole acetic acid (IAA) Mass production method. The method according to claim 1,
Further comprising the step of purifying the rooted seedlings obtained in said rooting step into soil. The method according to claim 1,
Wherein said disinfection comprises the steps of primary disinfection with a surfactant, washing with distilled water, secondary disinfection with alcohol, final disinfection with sodium hypochlorite and washing with distilled water. . The method according to claim 1,
Wherein the young eyes are cultured for 14 to 21 days in a shoot formation medium at 22 to 27 DEG C with light irradiation at 2000 to 3000 lux for 15 to 17 hours for the shoot formation. The method according to claim 1,
Wherein the shoot is cultured in seedling seed culture at 22-27 ° C at 2000-3000 lux for 15-17 hours with light irradiation for 28-35 days. The method according to claim 1,
Wherein the seedling is cultured in a seedling growth medium at 22 to 27 DEG C for 15 to 17 hours at 2000 to 3000 lux for 14 to 21 days to grow the seedling. The method according to claim 1,
Characterized in that the seedling is cultured in a rooting medium at 22-27 ° C in the range of 2000-3000 lux for 15-17 hours with light for 13-15 days.

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