KR20160092938A

KR20160092938A - Propagation Method of Vaccinium oldhami

Info

Publication number: KR20160092938A
Application number: KR1020160009885A
Authority: KR
Inventors: 곽명철; 채정우
Original assignee: 경기도
Priority date: 2015-01-28
Filing date: 2016-01-27
Publication date: 2016-08-05
Also published as: KR101852799B1

Abstract

The present invention provides a propagation method of pure gold trees. Also provided is a method for mass regeneration of Vaccinium oldhami. According to the present invention, the method of the present invention is a novel method which can replace the ginkgo tree propagation method which has been difficult to cultivate, propagate and manage, and can efficiently and continuously propagate a large amount of plants, Thereby contributing to stable maintenance and improved productivity.

Description

Propagation Method of Vaccinium oldhami}

The present invention relates to a method of breeding pure gold.

Tissue culture began to be attempted after the plant was found to have a totipotent ability to differentiate into individuals with some tissues (or cells). In plant tissue culture, seedling production generally regenerates small pieces of the fissional tissue of a representative plant under a sterile condition with a seed or shoot. The tissue culture test started with carrots was started in 1960s as a horticultural herb, and now it has been extended to carnation and lily as well as pine trees, acacia, and yukari. It is also recognized as an efficient propagation method for species that are difficult to reproduce by meteorological reproduction or conventional silent reproduction by plant propagation. Especially, tissue culture is used to propagate precious forest plants. Tissue culture is useful as a breeding method even in cases where mass multiplication is not difficult, and mass proliferation is necessary. In Korea, cultivation methods for rare forest plants are being studied, and the target species is diversified because the genetic traits are the same and the disease free hosts can be obtained.

Meanwhile, blueberries have been selected by the New York Times as the world's top 10 health food along with tomatoes, garlic, green tea, spinach, red wine, salmon, nuts, broccoli and oats. More than 250 species (including varieties) exist. Currently, all blueberries grown in Korea are imported from abroad.

There are blueberry varieties distributed in Korea. It belongs to the family Vaccinium and related species include the spruce tree ( Vaccinium bracteatum Thunb.), corn cherry ( Vaccinium There is hirtum . koreanum (Nakai) Kitam.), Vaccinium japonicum Miq., Vaccinium oldhamii Miq.), blueberry ( Vaccinium uliginosum L.), oranges ( Vaccinium vitis - idaea L.). Among them, the blueberry trees that grow in cold regions are widely distributed in North Korea, and the fruit is picked up from Baekdusan area and made into liquor and sold as "Korean blueberry wine or strawberry drink" It is distributed evenly, and it has been said that it has eaten liquor or edible in sugar with its fruit, and records can be found.

Other species are trees that grow into a small number of individuals in a specific area.

Recently, it has been reported that the research on the physiological action such as the antioxidative activity of the japanese tree, which is called Korean type blueberry, has a potency two to three times as high as that of the cultivated blueberry.

However, the reason why these pure gold trees can not be cultivated as a substitute for blueberries of foreign cultivars is that they can only be seen in nature and were not cultivated. In the case of many private cultivation attempts and past researches, it was found that when using the conventional method of cutting the blueberry as a cutting method, it was almost ineffective or less than 10%, and the seedling was sown and cultivated. There were no successful cases.

In other words, germination rate of seeds is low in seeds and it is difficult to propagate through the conventional propagation method (seeding, grafting, cutting).

The development of a new method for the efficient propagation of this kind of jade tree has been demanded and the success of the ginkgo tree propagation method using the tissue culture method is high in terms of the success of the gilding tree and the economical cultivation which is effective .

The inventors of the present invention have found that it is possible to use a plant tissue that uses plant leaves, roots, or other plant tissues in order to obtain a sufficient population through efficient propagation and mass proliferation in a short period of time in a jewelery tree which is difficult to breed, We have tried to develop a new method which can increase the embryogenesis and regeneration rate and mass production by using culture technology. As a result, the inventors of the present invention have determined the optimum medium, salt concentration, and pH (pH) suitable for germination of ginseng tree and induced callus capable of regeneration through growth organs, hormone type and concentration suitable for callus induction. In addition, more calli were induced through the wounding without cutting the root (root) in the cabin, and the types and concentrations of hormones suitable for proliferation were confirmed. In addition, the present invention has been accomplished by collecting fragments of ginkgo trees and directly inducing plant regeneration using appropriate hormone type, concentration, and light environment to grow into complete plants.

Accordingly, an object of the present invention is to provide an in-flight breeding method of pure gold.

Another object of the present invention is to provide a jade tree seedlings produced by the above-described method.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides an in-flight breeding method of Vaccinium oldhami comprising the following steps:

(a) Embryogenic calli from seeds, roots, branches, leaves or stems of Vaccinium oldhami are selected from the group consisting of Murashige and Skoog (MS), McCown Woody Plant Medium (WPM) and ADS Deriving from at least one medium;

(b) deriving the embryo or plant from the callus of step (a) in at least one medium selected from the group consisting of MS (Murashige and Skoog), WPM (McCown Woody Plant Medium) and ADS ; And

(c) purifying the plant by transplanting the plant of step (b) into artificial soil.

According to a preferred embodiment of the present invention, the medium of step (a) and step (b) is selected from the group consisting of Murashige and Skoog, WPM (McCown Woody Plant Medium) and ADS , More preferably a WPM (McCown Woody Plant Medium) or an ADS (Anderson) medium, and most preferably a WPM (McCown Woody Plant Medium) medium.

In addition, the medium includes 2-5% sucrose and 0.2-0.5% Gelrite.

According to a preferred embodiment of the present invention, the pH range of the medium of step (a) ranges from pH 4 to pH 6. [

According to a preferred embodiment of the present invention, the step (a) induces embryogenic calli by culturing for 4 to 8 weeks at a temperature of 22 to 25 ° C.

According to a preferred embodiment of the present invention, a suitable source for deriving embryogenic calli of step (a) is a seed or root of Vaccinium oldhami, more preferably a root.

According to a preferred embodiment of the present invention, the seed of step (a) is an immature seed or a ripened seed.

In the present invention, in-flight germination is preferable to a full-grown seed, and callus induction is preferably an immature seed. In addition, it is preferable to induce callus regeneration by WPM medium in which 0.5 mg / L of zeatin and 2,4-D are added at the root by scarring.

According to a preferred embodiment of the present invention, the roots are cut without cutting, and the medium composition is WPM, sucrose 3%, 2,4-D 0.5 mg / L, zeatin 0.5 mg / A pH of 5.3 is preferred.

According to a preferred embodiment of the present invention, the pH range of the medium of step (b) ranges from pH 5 to pH 5.5, most preferably pH 5.3.

According to a preferred embodiment of the present invention, the step (b) is cultivated at a temperature of 22 to 25 ° C for 4 to 6 weeks to induce embryo and plant formation.

According to a preferred embodiment of the present invention, the medium of step (a) or step (b) further comprises a growth regulator.

The term "growth regulator " as used herein means a chemical substance that regulates the growth and development of a plant, and includes a natural plant hormone produced in the body and a similar artificially produced synthetic hormone.

The growth regulator used in the present invention is auxin, gibberellin. Cytokinins, ethylene, gibberellin biosynthesis inhibitors, and the like, but are not limited thereto.

According to a preferred embodiment of the present invention, the growth regulator is selected from the group consisting of gibberellic acid (GA3), 2,4-dichlorophenoxy acetic acid (2,4-D), indole acetic acid (IAA) Indolebutyric acid (IBA), naphthaleneacetic acid (NAA), 4-CPA (Tomatotone), 2,4,5-TP (2,4,5-Trichlorophenoxypropionic acid), MCPA (2-methyl- but are not limited to, chlorophenoxyacetic acid, kinetin, zeatin, diphenylurea, abscisic acid (ABA), and BA (Benzyl Adenine) Do not.

According to a preferred embodiment of the present invention, the plant of step (c) uses at least one soil selected from the group consisting of vermiculite, pelrite and peatmoss.

In the step of inducing embryogenic callus from the seed of the present invention, the immature seed of the present invention is washed and surface sterilized according to a conventional manner prior to embryogenic callus induction. It may or may not injure immature seeds prior to embryogenic callus induction, which results in higher embryogenic callus induction efficiency when wounded.

The immature seeds can be cultured in a WPM (McCown Woody Plant Medium) medium at pH 4 to pH 6 containing 2-5% sucrose as a carbon source and 0.2-0.5% gelite as a hardening agent to induce embryogenic callus . The culture at this stage is maintained by incubation at a temperature of 22 to 25 ° C for 4 to 8 weeks.

Also, the WPM medium can control the concentration of the salt to 1/2 to 2 times.

In addition, a growth regulator may be added to the medium.

In the embryonic or plant-forming step from the callus of the present invention, the embryogenic callus is grown in a WPM (McCown Woody Plant Medium) medium at pH 5.3 containing 2-5% sucrose as a carbon source and 0.2-0.5% The embryo or plant may be induced by culturing. The culture at this stage is maintained at a temperature of 22 to 25 DEG C for 4 to 6 weeks. Also, the WPM medium can control the concentration of the salt to 1/2 to 2 times. In addition, a growth regulator may be added to the medium.

In the purifying step of the plant of the present invention, the embryo or plant may be purified by transplanting into soil containing one or more of vermiculite, pelrite and peatmoss. Preferably, the soil was purged into peatmoss, vermiculite, and pearlite soil mixed at a ratio of 1: 1: 1. After soil transplantation, the plants can be grown at a temperature of 22 to 25 ° C, a light intensity of 2200 to 3500 lux, 14 hours per day for 3-5 weeks in an incubation room of a photoperiod, and then seedlings capable of being transplanted into greenhouses and forages.

Each step of the in-flight propagation method will be described in detail as follows.

Step 1: In-flight germination stage in ripe seed

Seed Disinfection: Seedling seeds were immersed in tap water for 3 hours. Only seeds that had been submerged were selected, washed vigorously in water containing Tween 20, and washed several times with running water. The aseptic ash was transferred to a clean bench, sterilized with 70% ethanol for 1 minute and 30 seconds, and washed three times with sterilized water. Then, it was sterilized with 2% NaOCl solution for 5 minutes, rinsed with sterilized water more than 5 times, and dried for 30 minutes under a UV lamp in a clean bench.

Seed germination: MS (Murashige and Skoog), WPM (McCown Woody Plant Medium) and ADS (Anderson) powder media were used for germination experiments. Sucrose 3% was added to all three media and media were prepared at pH 4.5, 4.8, 5.0, 5.3 and 5.6 to determine titratable acidity. For solidification, 0.3% of gelrite was added and transferred to a Erlenmeyer flask. The flask was sterilized by a high pressure sterilizer at 121 ° C for 20 minutes and then solidified by placing it on a petri dish in a clean bench.

The seedlings were immature, mature seed (control) selected by submergence method, and immature and mature seeds treated with GA3 (100 ppm and 1,000 ppm) for 8 hours. The temperature of the culture room was 24 ± 2 ° C. and cultured in a dark room. Subculture was carried out in the same medium every 4 weeks.

Seed germination: In the immature seeds, germination started in the cabin after 40 days from the date of the first germination, and germination rate was about 50% overall. The lowest number of seeds germinated in MS medium and the germination rate of WPM medium and Anderson medium were similar, but WPM medium showed slightly germination rate. The seeds treated with 100 ppm of GA3 in the Anderson medium showed the highest germination rate of 93% and the germination rate of seeds treated with 1,000 ppm of GA3 decreased to about 30%. The pH was not correlated with germination of ginseng seeds. Rice seeds showed a constant germination rate regardless of GA3 treatment. The germination rate was 83% in the untreated Anderson medium and the average germination rate was 53% in MS medium, 66% in WPM medium and 61% in Anderson medium. As with the immature seeds, the pH of the ripened seeds was not influenced by germination. As a result of subculturing the germinated cultures with MS, WPM and Anderson medium, normal growth was not observed in MS medium, and the subculture of WPM medium grew most normally. Among them, 52.5% of ripened seeds and 41% of immature seeds were found to be the best growth at pH5.3.

Step 2: Immature seed And regeneration using roots of germinated individuals in ripe seeds Callus Judo

Callus induction from seeds: A medium containing growth regulators was prepared to induce somatic embryogenesis using seeds of pure wood. Sucrose3% and gelrite0.3% were used in MS, WPM and ADS medium as in germination. The growth regulators were 2,4-D 0.3, 0.5 and 1.0 mg / L in auxin alone medium. The medium containing auxin and cytokinin was 2,4-D 0.5 mg / L and BA 0.1, 0.3 , And 0.5 mg / L, respectively. The seedlings were cut vertically using a scalpel and mature seeds and immature seeds were placed vertically so that the cut surface reached the medium. The incubation temperature was 24 ± 2 ℃, and the same medium was prepared every 4 weeks and subcultured. The cells were cultured in a dark room for 12 weeks.

When 2,4-D alone was administered to immature seeds and ripe seeds to induce somatic embryos, more calli were induced than immature seeds in immature seeds. The highest amount of callus was produced in MS medium, which was not suitable for germination and growth, and induction rate was up to 30% in medium supplemented with 2,4-D 1.0 mg / L. In the case of the ripened seed, the correlation between the amount of hormone and the callus induction was unclear, and 20% of callus was induced in the Anderson medium, unlike the immature seed.

The medium containing the auxin (2,4-D) and the cytokinin (BA) also produced a large amount of callus in the immature seeds such as the medium treated with 2,4-D alone, A much higher amount of callus was induced. In addition, calli were induced in MS medium in the case of single treatment, whereas callus was induced in mixed treatment without any correlation. The most abundant medium was Anderson's medium supplemented with 2,4-D0.5 mg / L and BA 0.3 mg / L and showed an induction rate of 90%. Inoculated seeds were added with 2,4-D0.5 mg / L and BA 0.5 mg / L to induce a uniform induction rate on the medium.

Callus induction from roots: Callus induction using the growth organs resulted in a part of soft yellow callus rooting in the roots and inducing callus using the growing roots. Sucrosr 3% and gelrite 0.3% were added to the WPM powder medium and adjusted to Ph5.3. After 2,4-D 0.5mg / L and zeatin 0.5mg / L were added, And 10 individuals were sacrificed. The temperature of the culture room was 24 ± 2 ° C and incubated at the culture stand for 16 hours with light of 1,800 Lux per day.

Leaves, stems and roots induce callus induction. Solid green callus was induced in leaves and stems, whereas soft callus was induced in roots. As shown in the previous study, the hard-green callus showed no ability to regenerate. In order to induce the callus by using the soft yellow-yellow callus-induced roots, the root was cut without cutting to obtain 2,4-D 0.5 mg / L BA was added to the medium supplemented with 0.5 mg / L. After 7 days, soft yellow callus was induced and a larger amount of callus was induced than in the experimental group.

Step 3: From callus Stem regeneration induction step

Callus derived from roots was able to proliferate callus in sucrose 3%, 2,4-D 0.5 mg / L, zeatin 0.5 mg / L, pH 5.3 in WPM medium. Moisture stress was induced to induce the stem of the proliferated callus. The osmotic pressure was adjusted to 5% sucrose and 0.4% gelrite. At 4 weeks after subculture with osmotic pressure regulator, the callus periphery began to browse and stalks were formed in non - browning yellow callus. The resulting stem was rapidly grown in a red LED lamp, and the obtained stem was cut into mass and subcultured with a growth medium.

Step 4: From callus In the induced stem Multipurpose Stem induction

Stem cells from callus were cultured in medium supplemented with cytokinin, and then stem propagation experiments were carried out. The proliferation medium was adjusted to WPM, sucrose 3%, gelrite 0.3%, pH 5.3, and BA and zeatin in cytokinin were adjusted to 0, 0.3 and 0.5, respectively. , 1.0 and 2.0 mg / L, respectively. As a result, the highest number of stem was induced at 0.5 mg / L of BA (1.8), and then the stem number gradually decreased at high concentration. Zeatin produced more stem than BA in total, and 3.0 stalks were induced at 0.3 mg / L.

Step 5: Roots occur in the induced stem

The induced stem was cut and the root development experiment was conducted. The medium treated with 1 / 2WPM, sucrose 3%, agar 0.8% and pH 5.3 was treated with 0.5 mg / L of IAA, IBA and NAA, The cells were subcultured in 1 / 2WPM medium in which growth regulators were removed. As a result of cultivation, NAA showed the highest rooting rate as 55.5%, and treatment of NAA is effective for rooting of mildew. In addition, 0.2, 0.5, 1.0, and 1.5 mg / L were applied to study the proper concentration of NAA. Experimental results showed that the highest number of roots were induced at 1.0 mg / L of NAA.

Step 6: Plant purification

In the present invention, the embryo or plant may be purified by transplanting in a soil containing one or more of vermiculite, pelrite and peatmoss. Preferably, the soil was purged into peatmoss, vermiculite, and pearlite soil mixed at a ratio of 1: 1: 1.

After soil transplantation, the plants can be grown at a temperature of 22 to 25 ° C, a light intensity of 2200 to 3500 lux, 14 hours per day for 3-5 weeks in an incubation room of a photoperiod, and then seedlings capable of being transplanted into greenhouses and forages.

According to another aspect of the present invention, the present invention provides a method for mass regrowth of Vaccinium oldhami comprising the steps of:

(a) Incubating sections from seeds, roots, branches, leaves or stems of Vaccinium oldhami in a medium containing growth regulators, 2-5% sucrose and 0.2-1% agar Thereby inducing a root of the culture; And

(b) promoting the growth of the culture of step (a).

As used herein, the term "segment" refers to a cell, tissue or organ part of a gut tree (Vaccinium oldhami), as long as the segment can induce callus, embryo or root, Can be used.

Since the regeneration method of the present invention utilizes the above-described fragments of the ginseng tree, some of the steps may be the same or similar to the in-flight breeding method, and the steps may be arbitrarily controlled by the person skilled in the art to add, have.

According to a preferred embodiment of the present invention, the medium of step (a) is at least one selected from the group consisting of MS (Murashige and Skoog), WPM (McCown Woody Plant Medium) and ADS (Anderson) Is a WPM (McCown Woody Plant Medium) or ADS (Anderson) medium, and most preferably a 1 / 2WPM (McCown Woody Plant Medium) medium.

The pH range of the medium of the present invention ranges from pH 4 to pH 6, preferably pH 5.3.

In addition, the step (a) may induce roots under a condition of lighting (1,800 lux) at a temperature of 22 to 26 ° C for 16 hours per day.

According to a preferred embodiment of the present invention, the growth regulator is selected from the group consisting of gibberellic acid (GA3), 2,4-dichlorophenoxy acetic acid (2,4-D), indole acetic acid (IAA) Indolebutyric acid (IBA), naphthaleneacetic acid (NAA), 4-CPA (Tomatotone), 2,4,5-TP (2,4,5-Trichlorophenoxypropionic acid), MCPA (2-methyl- at least one member selected from the group consisting of chlorophenoxyacetic acid, kinetin, zeatin, diphenylurea, abscisic acid (ABA) and BA (Benzyl Adenine) Is indolebutyric acid (IBA) or naphthaleneacetic acid (NAA), and most preferably naphthaleneacetic acid (NAA).

According to a preferred embodiment of the present invention, the growth regulator is contained at 0.1 mg / L to 3 mg / L, more preferably 0.1 mg / L to 2 mg / L, and most preferably 1 mg / L .

In one embodiment of the present invention, when callus induction, 2,4-D, 0.5 mg / L + zeatin, 0.5 mg / L of the growth regulator was scratched to induce callus proliferation, callus proliferation to the same medium, + sucrose 5% + gelrite 0.4% + pH 5.3. In addition, stem growth is induced by adding 0.3% zeatin to the callus-derived stem, and roots induction is induced by subculturing the medium with 1.0 mg / L NAA supplemented with 1 / 2WPM medium for 2 weeks.

According to a preferred embodiment of the present invention, the light environment may be promoted in step (b) to promote growth, and the light environment may include irradiating the LED with light.

According to a preferred embodiment of the present invention, the LED is at least one selected from the group consisting of red light and white light, and is most preferably red light.

Each step of the mass regeneration method will be described in detail as follows.

Stage 1: inducing root formation from sections

Some sections of the cells, tissues or organs of the gill tree of the present invention include plant hormones such as naphthaleneacetic acid (NAA), 2-5% sucrose and 0.2-1% agar as growth regulators (McCown Woody Plant Medium) medium of pH 4 to pH 6 for 2 weeks to stimulate root development, followed by subculture with WPM (McCown Woody Plant Medium) medium containing no plant hormone.

The culture at this stage can be maintained at a temperature of 22 to 26 DEG C, a light intensity of 2200 to 3500 lux, and a culture period of 16 hours per day for 4 to 8 weeks.

Stage 2: Facilitating the growth of plant cultures

The plant cultures can promote maturation or growth using an optical environment. Preferably, the red light LED may be irradiated.

After regeneration with root formation and growth, the seedlings are transplanted into greenhouses and forages after being fired at a temperature of 22 to 25 ° C, a light intensity of 2200 to 3500 lux, 14 hours per day for 3 to 5 weeks in the incubation room of photoperiod Can be cultivated.

Therefore, the regeneration method of the mildew tree of the present invention directly regroups cells, tissues, or parts of organs of a plant and cultivates them into complete plants, so that the plants can be grown in a large amount in a short time.

In addition, the callus and shoot induction steps in conventional tissue culture are omitted, and the tissue culture period can be greatly shortened. By using the plant hormone of the present invention and the plant regeneration promoting method using specific light irradiation, it is possible to proliferate and produce a large amount of pure wood, so that the plant can be rapidly supplied at a desired time.

Since the regeneration method of the present invention uses the above-described pure gold tree, the common description thereof is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

In accordance with another aspect of the present invention, the present invention provides a jade tree seedlings produced by the methods of the present invention.

The method of the present invention is a new method that can replace the ginkgo tree propagation method which has difficulties in conventional cultivation, propagation and management, and can efficiently and continuously propagate a large amount of plants, thereby securing stable seedling and improving productivity . &Lt; / RTI >

Figure 1 shows the germination rate of immature seeds at each treatment.
Fig. 2 shows the seed germination rate of the ripe seeds according to the treatments.
Figure 3 shows the survival rates of immature and ripe seeds by media and pH.
Figure 4 shows the pure wood transformation rate.
Fig. 5 shows the process of cultivation and purification in a gilded tree. (A: immature seed germination, b: mature seed germination, c: germination in vitro, d: growth after passage, e: soil purification).
Figure 6 shows callus induction rates following 2,4-D hormone treatment.
FIG. 7 shows the callus induction rate of 2,4-D and BA hormone mixed treatment.
FIG. 8A shows a callus generated from a ginkgo tree seed, and FIG. 8B shows a callus induced from a wounded root. Fig. 8c shows the pure tree trunks produced by regeneration in the proliferated callus. FIG. 8d shows the occurrence of stems for each concentration of BA and zeatin.
Fig. 9 shows the regeneration of the ginseng callus (ab: callus-induced shoot, c: multi-shoot occurrence, d: shoot root circumference)
Fig. 10 shows the number of adventitious inductions by BA concentration.
FIG. 11 shows changes in the number of adolescents per BA processing period.
12 shows the adventitious child guidance (left: 1 week, right: 3 weeks).
Figure 13 shows the callus induced in ginkgo tree roots.
Fig. 14 shows the growth of ginseng tree callus (left: light culture, right: dark culture).
Fig. 15 shows the weight loss of medium callus proliferation medium for 4 weeks.
FIG. 16A shows a rooted root image of a treated root of NAA 0.5 mg / L; FIG. 16B shows a rooted root image of IBA 0.5 mg / L treated mildew; And Fig. 16 (c) shows a root rooting result graph.
FIG. 17 shows a graph of the roots rooting rate of the ginseng root for each NAA concentration.
Fig. 18 shows the rooting results of ginkgo tree (6 weeks) for each NAA concentration.
FIG. 19 shows the growth results (3 months) of the pure wood LED color scheme.
FIG. 20 shows the results of the red LED survey on the pure gold tree (right) and the white LED survey result (left).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. In-flight germination stage in ripe seeds

Seed harvesting

Seedlings of ginseng seeds (early seeds in early September and end of October in seedless seeds) were collected from the native areas of Anyeon-eup, Taean-gun, Chungcheongnam-do.

Seed sterilization for culture

The seeds of the ginseng seeds used in this study were immersed in tap water for 3 hours. Only the seeds that had been submerged were selected, washed vigorously in water containing Tween 20, and washed several times with running water. To ensure sterile materials, they were transferred to a clean bench, sterilized with 70% ethanol for 1 minute and 30 seconds, and washed three times with sterile water. Then, it was sterilized with 2% NaOCl solution for 5 minutes, rinsed with sterilized water more than 5 times, and dried for 30 minutes under a UV lamp in a clean bench.

Seed dentition

Germination experiments were carried out in the cabin to investigate the optimal germination environment of the low - germination gypsum trees. Murashige and Skoog (MS), McCown Woody Plant Medium (WPM) and ADS (Anderson) powder media were used for germination experiments. All three mediums were supplemented with sucrose 3% and the medium was prepared at pH 4.5, 4.8, 5.0, 5.3 and 5.6 to determine the titratable acidity of germination. For solidification, 0.3-0.4% of gelrite was added and transferred to a Erlenmeyer flask. The flask was sterilized by a high pressure sterilizer at 121 ° C for 20 minutes and then solidified by placing it on a petri dish on a clean bench.

Seedlings were treated with immature, mature seeds selected by immersion method and immature and mature seeds treated with GAp ₃ at 100 ppm (0.01 g / 100 ml) and 1,000 ppm (0.1 g / 100 ml) for 1 hour. Three teeth were implanted and ten were repeated. The temperature of the culture room was 24 ± 2 ° C. and cultured in a dark room. Subculture was carried out in the same medium every 4 weeks.

As a result, as shown in Fig. 1, in the case of immature seed, germination started in the cabin after 40 days from the date of the first germination, and germination rate was about 50% as a whole. The lowest number of seeds germinated in MS medium and the germination rate of WPM medium and Anderson medium were similar, but WPM medium showed slightly germination rate. The seeds treated with GA ₃ 100 ppm in Anderson medium showed the highest germination rate of 93% and the germination rate of seeds treated with GA ₃ 1,000 ppm decreased to around 30%. The pH was not correlated with germination of ginseng seeds.

As shown in Fig. 2, the ripened seed showed a constant germination rate regardless of the treatment with GA ₃ . The germination rate was 83% in the untreated Anderson medium and the average germination rate was 53% in MS medium, 66% in WPM medium and 61% in Anderson medium. As with the immature seeds, the pH of the ripened seeds was not influenced by germination.

As shown in FIG. 3, germinated individuals were subcultured with MS, WPM, and Anderson medium. As a result, normal growth did not occur in MS medium, and subculture with WPM medium grew most normally. Among them, the highest growth was observed at pH 5.3 at 52% of ripened seeds and 41% of immature seeds.

In addition, the germinated seedlings grown in the above-mentioned germinated seedlings were soil-purified to peatmoss, vermiculite, and pearlite soil mixed at a ratio of 1: 1: 1 without roughening. (2 repetitions) were performed for 50 individuals per box, and the light condition was maintained at 2500 lux or more for 14 hours (6 to 20 hours) to investigate the removal of dead bodies and the growth rate.

As shown in Fig. 4, the soil purification was carried out twice with 50 individuals on normally grown individuals. As a result, 92% of the primary purification and 82% of the secondary purification were 88%.

Example 2. Immature seed And regeneration using roots of germinated individuals in ripe seeds Callus Judo

From seed Callus Judo

A medium containing a growth regulator was prepared to induce somatic embryogenesis using seeds of pure wood. Sucrose 3% and gelrite 0.3% were used in MS, WPM and ADS medium as in germination. The growth regulators were 2,4-D 0.3, 0.5 and 1.0 mg / L in auxin alone medium. The medium containing auxin and cytokinin was 2,4-D 0.5 mg / L and BA 0.1, 0.3 , And 0.5 mg / L, respectively. The seedlings were cut vertically using a scalpel and mature seeds and immature seeds were placed vertically so that the cut surface reached the medium. The incubation temperature was 24 ± 2 ℃, and the same medium was prepared every 4 weeks and subcultured. The cells were cultured in a dark room for 12 weeks.

As a result, as shown in Fig. 6, when 2,4-D alone was administered to immature seeds and ripe seeds to induce somatic embryos, a larger amount of callus was induced in immature seeds than in ripened seeds. The highest amount of callus was produced in MS medium, which was not suitable for germination and growth, and induction rate was up to 30% in medium supplemented with 2,4-D 1.0 mg / L. In the case of the ripened seed, the correlation between the amount of hormone and the callus induction was unclear, and 20% of callus was induced in the Anderson medium, unlike the immature seed.

Further, as shown in Fig. 7, even in a medium in which auxin (2,4-D) and cytokinin (BA) are mixed, 2,4- Of callus was formed and the amount of callus was much higher than that of single treatment. In addition, calli were induced in MS medium in the case of single treatment, whereas callus was induced in mixed treatment without any correlation. The most abundant medium was Anderson's medium supplemented with 0.5 mg / L of 2,4-D and 0.3 mg / L of BA, and showed an induction rate of 90%. Immature seeds were uniformly high induction rate on medium supplemented with 0.5-mg / L of 2,4-D and 0.5 mg / L of BA.

From the roots Callus Judo

The induction of callus using the growth organ was induced by a part of soft yellow callus in the roots. Sucrosr 3% and gelrite 0.3% were added to the WPM powder medium and adjusted to Ph5.3. After 2,4-D 0.5mg / L and zeatin 0.5mg / L were added, And 10 individuals were sacrificed. The temperature of the culture room was 24 ± 2 ° C and incubated at the culture stand for 16 hours with light of 1,800 Lux per day.

From each leaf, stem, and root, the callus was induced. Solid green callus was induced in the leaves and stems, while soft callus was induced in the roots (Fig. 8b). In hard green callus, it does not show regeneration ability. As shown in the previous research, it was used to induce callus using soft-yellow callus-induced roots. BA was added to the medium supplemented with 0.5 mg / L. After 7 days, soft yellow callus was induced and a larger amount of callus was induced than in the experimental group.

Therefore, although callus induction and regeneration are possible in immature seeds, high quality callus was found to be root - derived callus.

Example 3. From callus Stem regeneration induction step

The root-derived calli were able to proliferate callus in 3% sucrose, 0.5 mg / L 2,4-D, 0.5 mg / L zeatin and pH 5.3 in WPM medium. Moisture stress was induced to induce the stem of the proliferated callus. The osmotic pressure was adjusted to 5% sucrose and 0.4% gelrite. At 4 weeks after subculture with osmotic pressure regulator, the callus periphery began to browse and stalks were formed in non - browning yellow callus. The resulting stem was rapidly grown in a red LED lamp, and the obtained stem was cut into mass and subcultured in a growth medium (FIG. 8C).

In other words, the root development was induced by treatment of 5% sucrose and 0.4% gelrite using roots - induced callus, and the osmotic pressure was adjusted and the callus was subjected to water stress to induce stem development.

Example 4. From callus In the induced stem Multipurpose Stem induction

Stem cells from callus were cultured in medium supplemented with cytokinin, and then stem propagation experiments were carried out. The proliferation medium was adjusted to WPM, sucrose 3%, gelrite 0.3%, pH 5.3, and BA and zeatin in cytokinin were adjusted to 0, 0.3 and 0.5, respectively. , 1.0 and 2.0 mg / L, respectively. As a result, the highest number of stem was induced at 0.5 mg / L of BA (1.8), and then the stem number gradually decreased at high concentration. Zeatin produced more stem than BA in total, and 3.0 stalks were most abundant at 0.3 mg / L (Fig. 8d).

In other words, it was appropriate to treat zeatin 0.3 mg / L for stem growth.

Example 5. Roots occur in induced stem

The induced stem was cut and the root development experiment was conducted. The medium treated with 1 / 2WPM, sucrose 3%, agar 0.8% and pH 5.3 was treated with 0.5 mg / L of IAA, IBA and NAA, The cells were subcultured in 1 / 2WPM medium in which growth regulators were removed. As a result of cultivation, NAA showed the highest rooting rate as 55.5%, and treatment of NAA is effective for rooting of mildew. In addition, 0.2, 0.5, 1.0, and 1.5 mg / L were applied to study the proper concentration of NAA. Experimental results showed that the highest number of roots were induced at 1.0 mg / L of NAA. The plant purification step was then carried out.

Example 6. From callus Shinshu Judo

The callus produced from the roots of ginseng germinated and propagating in the cabin was used as experimental material. In the in vitro culture and germination experiments, 5% sucrose and 0.5% gelrite were added to the WPM medium to control the osmotic pressure and sterilized in a high pressure sterilizer.

Callus derived from the original roots was grown in WPM medium for 6 weeks by adding 0.5 mg / L of 2,4-D and zeatin, respectively. After 6 weeks, 20 ~ 30 stalks were formed when the callus was browned due to water stress.

Therefore, according to the present invention, it is possible to induce embryogenic calli from seeds, to maintain and proliferate embryogenic callus, to induce complete plant through germination and purification process, in a pure wood tree having low seed germination rate and difficulty in propagation have.

Example 7. Purewood Identify optimal conditions for mass regeneration

The present inventors carried out rooting induction experiments using the stem of a pure wood germinated in the above-mentioned cabinet in order to massively regrow the pure wood using hormones. The medium was treated with Sucrose 3% in 1 / 2WPM medium and solidified with agar 0.8%. In the plant hormones, 0.5 mg / L of each of IAA, NAA and IBA was added, followed by 2 weeks of stimulation and subcultured with 1 / 2WPM control medium. Three sections of one culture bottle (CB400) were implanted and repeated three times. The culture environment was 24 ± 2 ° C and 16 hours light (1,800 lux) per day.

As shown in the graph of Fig. 16C, roots were not generated in medium containing no growth regulator and medium containing IAA, but roots were formed in medium treated with NAA and IBA, respectively (Figs. 16A and 16B). After 2 weeks of stimulation with 0.5 mg / L NAA and 1 / 2WPM control medium, roots started to develop from 2 weeks after subculture. Roots were formed for 5 weeks after the subculture and the final rooting rate was 55.5%. Green or yellow callus was also formed in the section of the NAA stimulated section, and some individuals were rooted in the callus. In the medium treated with IBA, roots started to develop from 3 weeks after passage and showed rooting rate of 22.2% after 5 weeks. Comparing the rooting conditions of NAA and IBA treatment, NAA showed a large amount of coarse root, while a small number of thin roots were produced during treatment with IBA, and the leaf turned red and the growth was not good.

Therefore, NAA was found to be suitable for the occurrence of gruel root.

In addition, the optimum concentration of NAA suitable for roots of ginseng root was confirmed.

First, 1 / 2WPM medium was treated with 3% Sucrose and solidified to 0.8% Agar to treat NAA at 0.2, 0.5, 1.0 and 1.5 mg / L (pH 5.3). Thirty individuals were randomly assigned to each experimental group and subcultured with 1 / 2WPM medium 2 weeks later. The culture environment is the same as above.

As a result, as shown in FIG. 17 and FIG. 18, the rooting rate gradually increased as the NAA concentration increased, and the rooting rate was 50% when NAA 1.0 mg / L was treated. Since the rooting rate was decreased at the later concentration, it was found that treatment with NAA 1.0 mg / L is most suitable. Table 1 below shows rooting root rooting rate (8 weeks) of ginseng tree by NAA concentration.

NAA 0.2 mg / L 0.5 mg / L 1.0 mg / L 1.5 mg / L 2weeks 6.6% 13.3% 23.3% 6.6% 4weeks 6.6% 23.3% 26.6% 20.0% 8weeks 13.3% 36.6% 50.0% 46.6%

In addition, cultivation conditions using color LEDs suitable for gypsum tree propagation were confirmed.

In the conventional method, tissue culture was carried out in a white fluorescent lamp, but the effect of different color wavelengths was investigated.

The culture environment was the same as above, and the LED colors were white, red, blue, red + blue. In order to prevent each color from affecting other plants, each of them was provided with a dark coat. Ten individuals were germinated in the seeds (total 40 individuals).

The medium was treated with Sucrose 3% gelrite 0.4% and the pH was adjusted to 5.3% in the WPM medium with the best growth environment. After 3 months of growth, stem length, root length, and number of leaves were examined to determine the best growth color.

As a result, as shown in Fig. 19, the most growth was observed in the pure woods grown in the red color of the pure wood tree LED coloring. Stem length was about 1.9 times, root was about 1.7 times, and leaf number was 1.5 times higher than those grown in white. On the other hand, the growth of blue color was suppressed rather than white color in the experimental group treated with red and blue.

Therefore, it was confirmed that the red LED is effective for the short - term growth of the gruel. Table 2 below shows the results of growth (3 months) of the pure wood coloring.

Red (red) Blue Red + blue (red + b lue ) White Stem length 8.3 ± 4.1 1.3 ± 0.6 4.1 ± 2.7 4.4 ± 1.1 Root length 4.1 ± 0.8 2.0 ± 0.8 3.1 ± 0.7 2.4 ± 0.6 Leaf (leaf number) 15.6 ± 8.2 2.6 ± 2.7 13.4 ± 7.5 10.1 ± 2.0

On the other hand, the results of purple tree trunk propagation using color LED were confirmed.

The medium was treated with WPM + sucrose 3% + zeatin 0.3% and adjusted to pH 5.3. The stems of the mildew cultivated in the cabin were cut into 2 cm lengths and laid on the medium. The stems were streaked by 3 individuals per culture bottle. After one month, the number and length of the induced stem were measured.

As a result, as shown in FIG. 20, the stem was induced 1.6 times larger than the white LED after 5 weeks from the teeth, and the average length was 2.7 times larger than that of the white LED.

In addition, not only the growth rate is excellent in red LED but also stem is more induced, so that it is effective to proliferate on red LED even when stem propagation is carried out. Table 3 below shows the results of each LED illumination of pure wood.

LED lighting Stem induction per section Stem length Remarks White 2.3 ± 1.3 0.6 ± 0.4 Red color 3.6 ± 1.6 1.6 ± 0.8

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

In-flight breeding methods of Vaccinium oldhami including the following steps:
(a) Embryogenic calli from seeds, roots, branches, leaves or stems of Vaccinium oldhami were seeded in a medium selected from the group consisting of Murashige and Skoog (MS), McCown Woody Plant Medium (WPM) and ADS At least one species of medium;
(b) deriving the embryo or plant from the callus of step (a) in at least one medium selected from the group consisting of MS (Murashige and Skoog), WPM (McCown Woody Plant Medium) and ADS (Anderson) medium; And
(c) purifying the plant by transplanting the plant of step (b) into artificial soil.

2. The method of claim 1, wherein the medium of step (a) and step (b) comprises 2-5% sucrose and 0.2-0.5% Gelrite.

The method according to claim 1, wherein the pH range of the medium of step (a) is in the range of pH 4 to pH 6.

The method according to claim 1, wherein the pH range of the medium of step (b) ranges from pH 5 to pH 5.5.

The method of claim 1, wherein the medium of step (a) or step (b) further comprises a growth regulator.

The growth regulator according to claim 5, wherein the growth regulator is selected from the group consisting of gibberellic acid (GA3), 2,4-dichlorophenoxy acetic acid (2,4-D), indole acetic acid (IAA), indolebutyric acid acid, IBA), naphthaleneacetic acid (NAA), 4-CPA (Tomatotone), 2,4,5-TP (2,4,5-Trichlorophenoxypropionic acid), MCPA (2-methyl-4-chlorophenoxyacetic acid) , At least one selected from the group consisting of kinetin, zeatin, diphenylurea, abscisic acid (ABA) and BA (Benzyl Adenine).

The method according to claim 1, wherein the plant of step (c) uses at least one soil selected from the group consisting of vermiculite, pelrite, and peatmoss.

The method according to claim 1, wherein the embryogenic callus is induced by culturing in the step (a) at a temperature of 22 to 25 캜 for 4 to 8 weeks.

The method according to claim 1, wherein the seed of step (a) is an immature seed or a mature seed.

10. The method of claim 9, wherein the seed is wounded.

The method according to claim 1, wherein the step (b) comprises culturing at a temperature of 22 to 25 ° C for 4 to 6 weeks to induce embryo and plant formation.

Mass regrowth method of Vaccinium oldhami including the following steps:
(a) Incubating sections from seeds, roots, branches, leaves or stems of Vaccinium oldhami in a medium containing growth regulators, 2-5% sucrose and 0.2-1% agar Thereby inducing a root of the culture; And
(b) promoting the growth of the culture of step (a).

13. The method according to claim 12, wherein the medium of step (a) is at least one selected from the group consisting of MS (Murashige and Skoog), WPM (McCown Woody Plant Medium) and ADS (Anderson) medium.

13. The method of claim 12, wherein the growth regulator is gibberellic acid, 2,4-dichlorophenoxy acetic acid, indole acetic acid, acid, IBA), naphthaleneacetic acid (NAA), 4-CPA (Tomatotone), 2,4,5-TP (2,4,5-Trichlorophenoxypropionic acid), MCPA (2-methyl-4-chlorophenoxyacetic acid) , At least one selected from the group consisting of kinetin, zeatin, diphenylurea, abscisic acid (ABA) and BA (Benzyl Adenine).

13. The method according to claim 12, wherein the growth regulator is included at 0.1 mg / L to 3 mg / L.

13. The method according to claim 12, wherein the LED is irradiated when the growth of the step (b) is promoted.

17. The method of claim 16, wherein the LED is at least one selected from the group consisting of red light and white light.