KR101415687B1 - Multiple Propagation Methods of in vitro Plantlets Derived from Node Cultures of Aronia using Tissue Culture Techniques - Google Patents

Abstract

The present invention relates to a method for mass proliferation of Aronia plants using tissue culture of stem nodes of Aronia plants. By applying the plant tissue culture technology to the mass propagation of Aronia seedlings, it is possible to contribute to increase the income of the farm household by early establishment of the high-value-added beef cattle cultivation by substituting the import substitution effect of the seedlings and the smooth farming of the good seedlings .

Description

[0002] The present invention relates to a method for mass proliferation of a stem from a stem culture of Aronia using a tissue culture technique,

The present invention relates to a high-efficiency mass propagation method of Aronia plants using Aronia tissue culture.

Aronia ( Aronia melanocarpa ) is a new breed of berry belonging to the cow family. Its anthocyanin, an antioxidant in fruit, contains more than 80 times of grapes and more than 5 times of blueberries. It is an expensive fruit that boats.

Since Aronia has been widely used as a berries for high-income crops, it has been used for cultivation since it has been used for cultivation. Currently, it has been cultivated as an actual seedling in Korea, and occasionally it has been used as a nutrition propagation method. However, Has been a limiting factor in proliferation since it requires a large amount of proliferation material. In addition, the cultivars were also cultivated in the wild, so it was possible to apply them to the wild, but it was possible to develop a hybrid between black, red and purple chalk berries (Viking, Nero, Aronimela, Aaron, Rejin, Rubina and Otum Magic). Therefore, there is a problem that practical breeding of interspecific hybrids is difficult to actual cultivation due to unevenness of later grains, and that the period of nutrition growth (about 4 years) is longer until fertilization.

On the other hand, in the case of the tissue culture method, if only a small number of mother-of-pearl can be secured, mass production can be carried out by in-flight production during the year. Therefore, when tissue culture method is used, it is possible to perform initial culture as a small amount of material, and once the material for propagation is obtained in the initial culture, it is possible to proliferate annually by using this material. Furthermore, in order to continuously supply these excellent varieties having excellent genetic qualities by preserving their genetic characteristics, nutrition reproduction using the tissue culture technique must be performed, and the shortening period (about 3 years) is advantageous.

However, it is difficult to develop these techniques because of the various difficulties such as the degradation of re-differentiation ability and the pollution problem in tissues.
(Patent Document 1) KR10-2000-0025820 A
(Patent Document 2) KR 10-2012-0034327 A

Therefore, it is an object of the present invention to provide a tissue culture method capable of mass proliferation in a short period of time, in which proliferation efficiency is remarkably high as compared to the conventional breeding of Aronia plants by conventional cutting, The purpose of this research is to establish a smooth supply system of good quality seedlings.

According to an aspect of the present invention, there is provided a method for growing an Arrancia plant comprising the steps of:

(S0) Pre-culture pretreatment step in which pesticide is sprayed before culturing Aronia mosquito, followed by spraying with 500-1,500 times of fertilizer and 500-1,500 times of physiologically active substance at a ratio of 1: 1;

(S1) culturing in a WPM (McCown WOODY PLANT MEDIUM) medium containing 0.5 to 2 mg / L of zitin, followed by cutting shoots of the pretreated Aronia stalks to form shoots;

(S2) subculturing the shoots in an MS (Murashige & Skoog MEDIUM) medium containing 3 to 5 mg / L of dimethylallylaminopurine (2iP) to induce and propagate multiple young shoots; And

(S3) the step of breeding and purifying the hyperplasia.

The tissue culture method used in the present invention is a method of aseptically culturing a cell or tissue section of a plant in a nutrient medium containing inorganic salts, sugar, vitamins, amino acids, hormones, etc., , Callus, which is a cell mass, is formed. These callus are transferred to a new medium and cultured to obtain a young plant, or a plant already existing in the plant is grown as a complete plant. In particular, as in the present invention, since the tissue culture method using the stem node induces the elongation of the eye in the stem node existing in the aronia, the seedlings are propagated. Therefore, the genetic uniformity of regenerated aronia The growth rate of the eyes is shortened more than two months and the more robust seedlings can be obtained at the early stage than the other Aronia tissue culture cultures.

In the present invention, since the pre-incubation pre-treatment step is important due to the characteristics of the woody flow, an original method of minimizing the contamination rate through the pretreatment step (S0) and the sterilization step (S1) Type and concentration, treatment timing and method, and decompression treatment process.

The present invention also relates to a method for producing a growth medium comprising steps of (S1) to (S3), wherein the amount of the basic medium for growth and the growth regulator, the amount of the basic medium for growth culture and the amount of growth regulator, It is the core of this technology to suggest creative ways to maximize culture efficiency, namely to apply different basic media and growth regulators for each stage of cultivation, and to develop a method that enables rooting and purification at the same time.

That is, in order to effectively mass-propagate the seedlings through the tissue culture of the Aronia plants, the present invention can be applied to the development of pre-culture pretreatment methods, the use of stem nodes, simplification of the culture medium (using a single basic culture medium for each culture step) (Cultivation of various cultivars in the same medium), improvement of the efficiency of the medium preparation, and economic medium composition (development of various culture methods for industrialization such as cultivation of various cultivars and simplification of cultivation steps in a single medium.

In the present invention, "basic medium" means a culture medium containing only the most basic substances necessary for culturing a plant. In the present invention, WPM (McCOWN WOODY PLANT MEDIUM) or MS (Murashige & Skoog MEDIUM) Was used as the primary culture medium. The culture medium was prepared by adding giitin, dimethylallylaminopurine, kinetin or indole-3-butyric acid (IBA) Lt; / RTI >

In the present invention, WPM (Lloyd & McCown Woody Plant Basal Salt Medium) is used as a basic medium in the step of forming shoots, and MS (Murashige & Skoog Medium, Mod. No. 4 Basal Salt Mixture) medium as a basic medium.

In the present invention, the pH of the basic medium is 5 to 6, preferably 5.2 to 5.6. When the pH of the medium is used in excess of 6, the growth of the plant may be delayed because it does not conform to the characteristics of aronia that grow well in the acidic state. When the pH is lower than 5, And calcium deficiency, which can result in necrosis of the growth point.

Hereinafter, a method of growing Aronia plants in detail will be described step by step.

In the present invention, a pretreatment process is performed before the culturing in the step (S0). In the pretreatment process, pesticides are sprayed before culturing, and 500-1,500 times of the fertilizer and 500-1,500 times of the physiologically active substance are mixed at a ratio of 1: 1 It is a process of spraying. Such a process may be repeated at least twice in a period of 3 to 7 days. However, any person skilled in the art may appropriately perform the process according to the kind of cultivar, culture period, kind of fertilizer and physiologically active substance Can be adjusted. This pre-culture preprocessing process can minimize the contamination rate.

In the present invention, the step (S1) includes cutting the aronia stem. In the step (S1) of the present invention, the length of 0.2 to 0.5 cm from the upper part of the stem of the stomach of the Aronia and the length of 1.2 to 1.8 cm, preferably 1.5 cm, which cuts the lower part of 1 to 1.3 cm from the node It is desirable to use stem nodes.

In the present invention, it is preferable that 0.05 to 0.15% of the electrodeposited agent of 4 to 6 times the volume of the truncated arnia stem cell is cultured in the culture medium for initial culture after truncation of the stromal nodule in step (S1) The sterilizing agent may be further added to the stem of the cutter, and sterilized by stirring with 15 to 25 minutes under reduced pressure 2 to 3 times using a vacuum pump.

Examples of the bactericide used in the present invention include sodium hypochlorite (NaOCl) or calcium hypochlorite (Ca (ClO) ₂ ), but the present invention is not limited thereto.

The electrodeposition agent itself has no drug effect, and when the pesticide is sprayed on the leaf or stem surface of the crop, the cohesive force of the water is destroyed, so that the chemical solution spreads uniformly on the surface of the plant, And the present invention is not limited to the type of the electrodeposition agent that is commonly used in the art.

The process may further include a step of surface sterilization by immersing the truncated arnojima stem in 70% ethyl alcohol for 20 to 40 seconds before the step sterilization.

In the present invention, the young shoot formation step (S1) uses WPM (McCOWN WOODY PLANT MEDIUM) medium as a basic medium and zeatin is added thereto. 2 mg / L.

The zitin is a cytokinin-like plant growth hormone such as benzylaminopurine, Thidiazuron, kinetin, and dimethylallylaminopurine (2iP) Which is a substance that induces the formation of a significant difference in the growth response depending on the species. As can be seen from Test Example 2-2, it was found that the survival rate was remarkably superior to the case of adding dimethylallylaminopurine (2iP), which is one of the cytokines such as zitin, with ziatin. In addition, zitin contains 0.5 to 2 mg / L, preferably 0.5 to 1 mg / L. Addition of more than 2 ㎎ / L of giatine causes problems such as the appearance of defective newborn due to the early multiple branching phenomenon, and when added in an amount less than 0.5 ㎎ / L, do.

Preferably, the WPM medium may be supplemented with giatinic sucrose, zeatin, and agar added thereto. Specifically, WPM (McCown WOODY PLANT MEDIUM) 2 to 2.5 g / L, sucrose 10 to 30 g / 0.5 to 2 mg / L of tin and 6 to 10 g / L of agar. More preferably, the ziatin may comprise 0.5 to 1 mg / L.

When sucrose is more than 30 g / L, there is a problem in that the shoot is not faded or elongated due to the reverse osmosis depending on the subculture period, and when it is added in an amount less than 10 g / L, the growth of shoots is slowed down. When the agar is added in an amount exceeding 10 g / L, the elongation of the shoot is decreased, or when the agar is added in an amount less than 6 g / L, the shoot is not supported on the medium.

Further, at the time of shoot formation, the stem of the truncated stem can be formally cultured at a temperature of 25 ± 1 ° C at a slope of 20 to 30 °, preferably 25 °, with the front surface facing the front.

In the present invention, the induction of the multi young shoot in the step (S2) is carried out by using MS (Murashige & Skoog MEDIUM) as the basic medium, adding dimethylallylaminopurine thereto, and the dimethylallylaminopurine is 3 To 5 mg / L. More preferably 4 mg / L.

Compared with the case of adding dimethylallylaminopurine (2iP) and gatine, which is one of the cytokines, the addition of dimethylallylaminopurine showed a remarkably excellent growth rate. When dimethyl allyl aminopurine is added in an amount exceeding 5 mg / L, the production rate of defective seedlings is increased due to excessive branching. When the amount of dimethyl allyl aminopurine is less than 3 mg / L, There is a problem that the rate of formation of the soda is lowered.

Further, kaenetin, benzyladenine or a mixture thereof may be further included, and synergistic effect with dimethylallylaminopurine is exhibited, so that the growth rate is remarkably improved. On the contrary, when benzyladenine (BA) is included together with dimethylallylaminopurine, synergistic effect is exerted, but there is a problem of vitrification. The carinethine, benzyladenine or a mixture thereof may further contain 0.05 mg / L to 0.15 mg / L, more preferably 0.1 mg / L. When the amount of carbinase, benzyladenine or a mixture thereof is added in an amount exceeding 0.05 mg / L, there is a problem that the rate of modification is increased. When the amount is less than 0.15 mg / L, have.

In addition, preferably, a medium for proliferation in which dimethylarylaminopurine (2iP) other than sucrose and agar is additionally added to the MS medium can be used, and more preferably, a growth medium for proliferation which further contains kainethine, benzyladenine, A medium can be used. Specifically, proliferation including 4 to 4.5 g / L of MS (Murashige & Skoog MEDIUM) containing vitamins, 15 to 25 g / L of sucrose, 3 to 5 mg / L of dimethylallyl aminopurine and 7 to 8 g / , And preferably the dimethylallylaminopurine can be 4 mg / L. More preferably, it is more preferable that the composition comprises 4 to 4.5 g / L of MS (Murashige & Skoog MEDIUM), 15 to 25 g / L of sucrose, 3 to 5 mg / L of dimethylallylaminopurine, 7 to 8 g / Adenine or a mixture thereof may be subcultured in a growth medium containing 0.05 to 0.15 mg / L, and still more preferably the kineticin, benzyladenine or a mixture thereof may be 0.1 mg / L. Most preferably, it is most preferred that the composition comprises 4 to 4.5 g / L of MS (Murashige & Skoog MEDIUM), 15 to 25 g / L of sucrose, 4 mg / L of dimethylallylaminopurine, 7 to 8 g / L of agar, These mixtures can be subcultured in a growth medium containing 0.1 mg / L.

Preferably, the hyperhidrosis induction is cultured at 25 占 1 占 폚 and then subcultured every 6 to 8 weeks.

In the present invention, in step (S3), rooting and refinement are performed for multiple seconds.

In one embodiment, the rooting and refinement may be carried out in a cabin and followed by two further steps of re-crystallization outside the air, wherein root induction of the root can be accomplished in a rooting medium in the cabin. The re-hydration may be carried out by transferring the formed plant to soil, preferably soil, more preferably planting soil for sowing.

Specifically, roots induction can be carried out by culturing in a rooting medium containing indole-3-butyric acid (IBA) in MS medium. The indole-3-butyric acid preferably contains 0.5 mg / L or less, more preferably 1.5 to 0.5 mg / L, and most preferably 0.1 mg / L. If the amount of indole-3-butyric acid added is more than 0.5 mg / L, rooting is inhibited. If not, rooting is delayed. In one embodiment of the present invention, when 1 mg / L indole-3-butyric acid was added to a medium in which giatin was excluded during rooting induction, rooting was induced in the multiple shoots and the plant was formed .

In addition, preferably, a medium containing the indole-3-butyric acid and sucrose and agar in addition to the MS medium can be used. Specifically, 4 to 4.5 g / L of MS (Murashige & Skoog MEDIUM) containing vitamins, sucrose 25 In a rooting medium containing 35 g / L of indole-3-butyric acid (IBA) of 0.5 mg / L or less and 7 to 8 g / L of agar. More preferably 1.5 to 0.5 mg / L, and most preferably 0.1 mg / L.

At this time, it is preferably cultivated at 25 ± 1 ° C.

Alternatively, in another aspect, out-of-bed rooting and purification may be carried out in one step at the same time, without culturing for inducing rooting in the rooting medium. For example, it is possible to transplant outgrown seedlings into a seedling bed filled with culture soil in a separate purification chamber, so that the rooting can be performed simultaneously with the purification. Thus, the shortening of the seedling production period by the simplification of the cultivation step and the economical effect thereof are exerted.

In the present invention, the soil is characterized by a mixture of peat moss and pearlite. The soil is mixed with peat moss at a volume ratio of 1 to 3 times per day.

Peat moss is made of sphagnum moss that has been trapped in a layer of water for a long time and is airtight, partially carbonized, which does not decompose completely, so large cell structures can absorb water and air. It can be used as slowly and as long as necessary.

Pearlite is a volcanically produced perlite which is expanded at a high temperature of 760 ~ 1200 ℃ up to 4 ~ 12 times its original volume. It has sufficient weather and void content, it can help grow plants by forming air passages that are light in weight and provide optimum ventilation and drainage.

Therefore, when the pearlite is added to the peat moss in the soil of the present invention in an amount of 3 times or more by volume, the pore of the soil is increased, and the difference of the dry and wet conditions is large, which may adversely affect the growth of young seedlings. The water holding capacity of the soil is so high that the in-situ stems due to the humidification may decay, resulting in a marked reduction in the rate of exhumation of plants. Therefore, pearlite can be mixed with peat moss in a volume ratio of 0.5 to 3 times, and it is most preferable to mix pearlite with pearlite.

In the present invention, the above-mentioned Arrioni species and its modified varieties, such as Nero, Viking, Aronimela, Aron, Hugin and Rubina varieties, And two or more varieties selected from the group consisting of Aronia varieties. According to the present invention, the above-mentioned two or more varieties of Aronia can be cultured simultaneously in a single medium. Conventionally, when two or more varieties of cultivars are cultivated, the cultivars of Aronia were cultivated in separate culture media. However, in the culture medium according to the present invention, cultivars of two or more cultivars were cultured in a single culture medium Can also be cultured at an efficiency equal to or better than that of each cultured in a separate medium.

By applying the plant tissue culture technology, which is the state-of-art technology of seedling propagation according to the present invention, to the mass propagation of Aronia seedlings, it is possible to produce seedlings by replacing seeds with imported seeds, Settlement can contribute to increase farm household income. In addition, the cost of seedlings production period and personnel expenses have been reduced, and it is expected to create various effects such as the possibility of exploring export market of new crops by improving international competitiveness by improving the quality of products.

Figure 1 is a photograph of Aronia saponins and fruit (fruit).
2 is a photograph of shoots used as a material.
Fig. 3 is a photograph showing shoots formed through the cultivation of Aronia stalk node.
Figure 4 is a photograph of Aronia plants transplanted (subcultured) for hyperplasia.
FIG. 5 is a photograph of the Arrondia plant which has undergone the hyperplasia.
Fig. 6 is a photograph of an initial plant of Aronia inflated.
Fig. 7 is a photograph of a plant of Aronia that has been mobilized after the transplantation.
Fig. 8 is a photograph of a plant of Aronia transferred to a flowerpot.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Pre-culture sterilization step

Aronia melanocarpa, black choke berry) progenitor stem segments of one kind and gaeryangjong 'Viking' (cv Viking), 'nero' (cv Nero) and "Oh Ronnie Mela '(cv Aronimela) 3 varieties of cultured.

From April to May, we picked a clean healthy poultry that was not diseased and not infected with virus among the mothers cultivated in the greenhouse or vinyl house and managed the mothers two weeks before the incubation. 1,000 times of fertilizer (Hyponex N: P: K = 5: 10: 5) and 1,000 times of physiologically active substance (hypotonic) 1,000 days of the pesticide The drainage was mixed at 1: 1 and sprayed. This process was repeated twice in a 5-day cycle.

Then, cut off the upper 3 ~ 4 segments of the upper part of the shoots (April to October) or the 9 ~ 10 month of the half-cut leaves, cut it to a length of about 10cm, remove the leaves, wash well with tap water, And cut to a length of about 5 cm to facilitate sterilization.

Figure 1 is a photograph of Aronia saponins and fruit (fruit), and Figure 2 is a photograph of elongated shoots used as a culture material.

After sterilization, sterilized beakers were placed in sterilized beakers, and 70% ethyl alcohol was poured. After 30 seconds of immersion, the surfaces were sterilized, washed once with sterilized water, and then transferred to a filtering flask After adding 1% sodium hypochlorite (NaOCl) solution (0.1% of the electrodeposition agent), add 5 times or more of the volume of the material and sterilize by agitating in a stirrer for 20 minutes under reduced pressure 2-3 times with a vacuum pump.

< Test Example  1> Effect of pretreatment before culture on contamination of culture slice

In the same manner as in Example 1, the contamination rate was compared between the case where the pretreatment was performed before the culture and the case where the pretreatment was not performed. That is, from April to May, we picked clean healthy stocks from the greenhouses or greenhouses grown in plastic houses. In Group 1, 1,000 pints of fertilizer (Hyponex N: P: K = 5: 10: 5) and 1,000 times of physiological activity The process of spraying 1,000 times of the material (Hy-tonic) at 1: 1 was repeated twice in 5-day cycle. And group 2 did not undergo pre - culture pretreatment and compared contamination rates with group 1. As a result, as shown in Table 1, it was confirmed that the group 2 was slightly contaminated as compared with the group 1 that had undergone the pretreatment before culturing.

Pretreatment Whether or not to process Processed Do not process pollution - ++

Degree of pollution; - not, ++ somewhat polluted

< Example  2> The cultivation of nodes  through Shinshu  Forming step

The sterilized material was sterilized by flame sterilized sharp flower scissors. The damaged tissue (about 1 ~ 2mm) was removed, and then the remaining tissue was removed from the upper part of the embryo by about 2mm. About 1.5 cm in length) was used as a culture material.

The embryos were fixed in a glass test tube (25 mm in diameter and 150 mm in length) with 10 ml each of the initial culture medium as shown in the following Table 2 at a slope of about 25 ° with the front surface facing the culture chamber of 25 ± 1 ° C (1,500 lux, 16 hours, 8 hours).

Medium component  Amount added (in 1L) WPM containing vitamins
(McCOWN WOODY PLANT MEDIUM) 2.4 g Sucrose 20g Zeatin riboside &lt; RTI ID = 0.0 &gt; 0.5 to 1 mg Baby 7 ~ 8g pH 5.2 to 5.6

Fig. 3 is a photograph showing shoots formed through the cultivation of Aronia stalk node.

< Test Example  2-1> In the initial culture, After incubation  Effect on survival rate

Three different types of primary culture media were used for the initial culture. That is, 1 / 2MS containing 1/2 of ammonia nitrogen was used as a basic medium for WPM (McCown WOODY PLANT MEDIUM), MS (Murashige & Skoog MEDIUM) and MS medium, 2 in the same condition. As a result, as shown in Table 3, it was found that the survival rate was superior to the other media when WPM was used as the primary culture medium.

badge WPM MS 1 / 2MS survival ++++ +++ +++

Survival level: +++ Good, ++++ Very good

< Test Example  2-2> Basis for initial culture In the badge  The addition of growth regulator After incubation  Effect on survival rate

The effect of different growth regulators on the survival rate after culture with Aronia was investigated. The WPM medium was used as the primary medium, and all other conditions except the growth regulator were tested in the same manner as in Example 2.

As a result, compared with the case of adding 2ip of the same amount belonging to the cytokinins, the survival rate after the culture with Aronia was excellent in the case of adding giatin, and the survival rate was remarkably excellent at 0.5-2 mg / L, especially 0.5-1 mg / L .

Kinds Glyatin (mg / L) 2ip (mg / L) Concentration 0 0.2 0.5 One 2 4 0.5 One 2 4 survival + ++ ++++ ++++ +++ ++ ++ ++ ++ +

Survival level: + Bad, ++ Normal, +++ Good, ++++ Very good

< Example  3> Rechincho  Propagation step through induction step

After 2 weeks of incubation, shoots grow 2 ~ 3 times from the shoots. After 4 weeks, 5 ~ 6cm of newborns are elongated. In this case, 100ml of transparent plastic culture bottle having a diameter of 8 cm and a height of about 10 cm (Table 5) were divided, sterilized and solidified. The stem was cut so that two to three embryos were contained in the medium. Then, the leaves were removed, (1500 lux, 16 hours, 8 hours) in a culture room at 25 ± 1 ° C. After that, the seedlings were cultured in the same manner every 6 to 8 weeks, and the propagation of the seedlings was continued.

Medium component  Amount added (in 1L) MS containing vitamins (Murashige & Skoog MEDIUM)   4.4 g Sucrose   20g 2iP   4 mg Kaenetine, BA or a mixture thereof   0.1 mg Baby   7 ~ 8g pH 5.2 to 5.6

Fig. 4 is a photograph of the Arrondia plant in which hyperplasia is induced.

< Test Example  3-1> During proliferation  The type of basic medium is Subculture  Effect on growth and growth of intercept

The effects of different culture media on growth and growth were investigated in three different cultivars. That is, 1 / 2MS containing 1/2 of ammonia nitrogen was used as a basic medium for WPM (McCown WOODY PLANT MEDIUM), MS (Murashige & Skoog MEDIUM) and MS medium, 3. As a result, as shown in Table 6, when the MS was used as the primary culture medium, the growth and the growth rate were superior to those of the other media.

Basic badge WPM MS 1 / 2MS growth +++ ++++ +++

Growth degree: +++ Good, ++++ Very good

< Test Example  3-2> During proliferation  basic In the badge  Growth regulator Dye  Or Mixed Addition on the Growth and Growth of Subculture Slices

The effects of different growth regulators on growth and proliferation were investigated. The MS medium was used as the primary medium, and all of the conditions other than the growth regulator were tested in the same manner as in Example 3.

As a result, when 2ip was added, the growth rate and growth rate after incubation were generally better than those with the same amount of zitin. In the case of mixing 2ip with BA or kinetin, a particularly excellent growth was observed when 2ip of 4mg / L and BA or kinetin of 0.1mg / L were mixed. However, when BA and 2ip are mixed, there is a problem that they are vitrified, and production of healthy seedlings is difficult.

< Example  4-1> onboard Induction of rooting  And purification (2 step culture ; Cabin Rooting  And Outdoors  After implantation, purification)

After 6 ~ 8 weeks from the last passage, 5 ~ 6cm of growth can be obtained. These shoots are cut into 5cm length and cut into 100ml of medium (Table 8) in a transparent plastic culture bottle having a diameter of 8cm and a height of about 10cm The stem was cut into 5 cm lengths so that the growth point was included in the dispensed, sterilized, and solidified medium. Then, the stem was transplanted (subculture) by 10 individuals and cultured in the incubation room at 25 ± 1 ° C for 3-4,000 lux, Time rock) to induce rooting. 5 is a photograph of Aronia plants in which cabbage rooting is induced.

(3,000 lux), humidity (80%), temperature (25 ± 2 ℃), and CO (2) were applied on a 200-well plug tray containing seedling for planting after washing the agar with the tap water. ₂ Concentrations were automatically controlled and transferred to an automated purification chamber where fertilization and fertilization were carried out for 7 to 10 days after the outflow and purification process, and then transferred to a greenhouse for growth. At this time, the stem of the lower part which was short in length or remained after cutting was reused as propagation material. FIG. 7 is a photograph of Aronia plants that have been mobilized after out-of-flight transplantation, and FIG. 8 is a photograph of Aronia plants transferred to flower pots.

Medium component  Amount added (in 1L) MS containing vitamins (all other than NH ₄ NO ₃ half volume)   4.4 g Sucrose   30g IBA   1 mg Baby   7 ~ 8g                         pH 5.2 to 5.6

< Test Example  4-1> Induction of rooting  When the culture medium contains the basic medium Subculture  Intercept To rooting  Impact

The effects of roots on roostering were investigated by three kinds of basic media used for roots induction culture. That is, 1 / 2MS containing 1/2 of ammonia nitrogen was used as a basic medium for WPM (McCown WOODY PLANT MEDIUM), MS (Murashige & Skoog MEDIUM) and MS medium, 4-1. As a result, as can be seen from Table 9, when the MS medium was used as the basic medium for proliferation, the rooting rate was excellent as compared with the WPM medium. Especially, MS medium containing ½ ammonia nitrogen showed excellent rooting degree equivalent to that of MS medium. In addition, when 1 / 2MS was used as a primary culture medium, it showed excellent logistic and rhizomes compared with MS medium.

badge MS MS (HN ₄ NO ₃ half) WPM Rooting ++++ ++++ +++

Rootability: +++ Good, ++++ Very good

< Test Example  4-2> Induction of rooting  When cultured, IBA The addition of To rooting  Impact

The effects of different growth regulators on root growth were investigated. The 1 / 2MS medium was used as the primary medium, and all the conditions other than the IBA concentration were tested in the same manner as in Example 4-1.

The results showed that the addition of 0.5 mg / L IBA resulted in a good rooting rate, especially when 0.1 mg / L IBA was added. Even though IBA was not added, it showed good rooting rate. However, there was a disadvantage that the number of days required for rooting was longer than that of IBA.

density 0 0.1 0.5 One Rooting +++ ++++ +++ ++

Rooting rate: ++ normal, +++ good, ++++ very good

< Example  4-2> Outdoors Induction of rooting  And purification (1 step culure ; Rooting  And purification steps)

After 6 ~ 8 weeks from the last subculture for the purpose of proliferation, 5 ~ 6cm of fresh shoots can be obtained. These shoots are cut into 5cm lengths and transferred to the rooting induction medium (3,000 lux), humidity (80%), temperature (25 ± 2 ° C) and CO ₂ Concentration was automatically controlled and fertilized by transferring the watering and fertilizer to an automated purification chamber for 7 ~ 10 days. Fig. 6 is a photograph of an out-of-flight transplanted Aronia plant.

By applying the plant tissue culture technology, which is the state-of-art technology of seedling propagation according to the present invention, to the mass propagation of Aronia seedlings, it is possible to produce seedlings by replacing seeds with imported seeds, Settlement can contribute to increase farm household income. In addition, the cost of seedlings production is greatly reduced, labor costs are reduced, and international competitiveness can be improved by improving the quality of products.

Claims (13)

A method for growing an Arrhia plant comprising the steps of:
(S0) Pre-culture pretreatment step in which pesticide is sprayed before culturing Aronia corn, and 500 to 1500 times of fertilizer and 500 to 1500 times of physiologically active substance are mixed at 1: 1 respectively and sprayed;
(S1) culturing in a WPM (McCown WOODY PLANT MEDIUM) medium containing 0.5 to 2 mg / L of zitin, followed by cutting shoots of the pretreated Aronia stalks to form shoots;
(S2) subculturing the shoots in an MS (Murashige & Skoog MEDIUM) medium containing 3 to 5 mg / L of dimethylallylaminopurine (2iP) to induce and propagate multiple young shoots; And
(S3) the step of breeding and purifying the hyperplasia. The method according to claim 1, wherein the step (S0) is repeated two or more times at a period of 3-7 days. 2. The method according to claim 1, wherein in step (S1), 0.2 to 0.5 cm from the upper part of the Aronia stem node ear and 1 to 1.3 cm from the lower part are cut to obtain stem nodes. 2. The method according to claim 1, wherein the sterilizing agent containing 0.05 to 0.15% of the electrodeposited agent, which is 4 to 6 times the volume of the truncated arniae sternum in step (S1), is applied to the truncated arnia stem, And sterilized by stirring for 15 to 25 minutes while being decompressed 2 to 3 times. 2. The method according to claim 1, wherein in step (S1), 2 to 2.5 g / L of WPM containing McCOWN WOODY PLANT MEDIUM, 10 to 30 g / L of sucrose, 0.5 to 2 mg / ) &Lt; / RTI &gt; 6 to 10 g / L. The method according to claim 1, wherein in step (S2), 4 to 4.5 g / L of MS (Murashige & Skoog MEDIUM) containing vitamins, 15 to 25 g / L of sucrose, 7 to 8 g / (2 &lt; i &gt; P) in an amount of 3 to 5 mg / L.  7. The method according to any one of claims 1 to 6, wherein the medium in step (S2) further comprises 0.05 to 0.15 mg / L of kinetin, benzyladenine or a mixture thereof. [3] The method according to claim 1, wherein, in step (S3), rooting of multiple seconds is induced by culturing hypericin in MS medium containing 0.5 mg / L or less of indole-3-butyric acid &Lt; / RTI &gt; 9. The method according to claim 8, wherein in step (S3), 4 to 4.5 g / L of MS containing vitamin, 25 to 35 g / L of sucrose, 0.5 mg / L or less of indole- &Lt; / RTI &gt; to 8 g / L. The method according to claim 8 or 9, wherein the step (S3) comprises 0.1 mg / L of indole-3-butyric acid. 9. The method of claim 8, further comprising the step of inducing and purifying rooting in the cabin and then recirculating outside. 2. The method according to claim 1, wherein step (S3) is performed at the same time outside the planting and refinement. The method according to claim 1, further comprising the steps of: contacting Nero, Aronia Viking, Aronimela, Aron, Hugin and Rubina, Wherein at least two cultivars selected from the group consisting of the cultivars of Aronia are cultivated simultaneously in a single medium.

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