KR100767050B1 - Somatic Embryogenesis and Mass propagation of Kalopanax septemlobus Thunb. Koidz without thorns by in vitro culture - Google Patents

Abstract

The present invention relates to a mass production method through in-flight culture of Kalopanax septemlobus (Thunb. Koidz.) Seedlings.

The present invention is a step of inducing germ cells and callus aseptically from the leaf disease of plants, inducing somatic embryos from embryogenic callus, and inducing and growing seedlings through the development process from induced somatic embryos And inducing and propagating secondary embryogenic callus using cultured in-flight plants, and inducing a large amount of secondary somatic embryos through liquid and solid medium from the induced secondary embryogenic callus; The present invention relates to a method for mass production of thorny seedling seedlings comprising the step of producing a large amount of seedlings from somatic embryos, and purifying the seedlings from which the seedlings were grown.

The present invention can produce a large amount of seedlings of thorny trees that are difficult to harvest conventional seeds, difficult to reproduce in real time, and can produce a large amount of seedlings of the trees through efficient soil purification of the plants.

Description

Somatic Embryogenesis and Mass Propagation of Kalopanax septemlobus (Thunb.) Koidz without thorns by in vitro culture}             

1 is a photograph showing the primary callus induced from the leaf disease.

Figure 2 is a photograph showing the primary embryogenic callus proliferation.

Figure 3 is a photograph showing the primary somatic embryos derived from primary embryogenic callus.

Figure 4 is a photograph showing a seedling derived from the primary somatic embryo.

5 is a photograph showing the in-plant plants in which the primary seedlings are grown.

Fig. 6 is a photograph showing the plant slices and induced callus in the first cabin.

a) Photograph showing the plant section slices and induced callus in the first cabin.

b) A picture showing callus derived from plant root slices in the first cabin.

c) Photograph showing callus derived from primary somatic embryos and slices of plant leaves, roots and leaf disease on board.

7 is a photograph showing secondary callus proliferation induced from primary plant sections.

a) Photograph showing secondary callus proliferation induced from root sections.

b) Secondary callus proliferation induced from leaf sections.

Figure 8 is a photograph showing the second embryogenic callus and embryogenic callus derived somatic embryos

a) Picture showing embryogenic callus derived from primary plant sections.

b) Photograph showing secondary somatic embryos derived from secondary embryogenic callus.

Figure 9 is a photograph showing the somatic embryo and seedling induction and growth through liquid culture.

10 is a photograph showing a plant body propagated through secondary somatic embryos.

11 is a photograph showing the purification of the plant in the cabin.

The present invention relates to a method for inducing germ cells capable of inducing plants from the tissues of Kalopanax septemlobus (Thunb. Koidz.) And culturing these cells to produce plants in large quantities in a culture vessel. .

In particular, the present invention induces the seedlings by inducing callus after inducing callus from leafy leaves of the thorny tree, and continuously inducing embryogenic callus through the seedlings, and then inducing secondary somatic embryos to germinate. After cultivating the seedlings and soil cultivation of the in-flight plants to a method of mass production of thorny seedling seedlings.

Yin-tree is a deciduous broad-leaved arborescent tree belonging to the arboraceae family, distributed throughout the country's wild fields, and it is known to contain Liriodendrin, Hederagenin, Arabinose, Lignan, Kalotoxin, and Kalosaponin.

In Chinese medicine, the herbal medicines of Yin-mung are called Haedong-mok and Ha-ja-mok-mok, which are used as medicines for cancer, diabetes, chronic hepatitis, chronic colitis, liver disease, pleurisy, edema, and chronic gastritis. It is known to have various effects such as preventing.

In addition, Yin-myeon is used in wild springs with its high taste and fragrance.

There have been no reports on the tissue culture of thorny trees. Studies on the tissue culture of elmaceae plants, such as elm, have been shown to induce plants by inducing secondary folds directly from the first immature embryos (Gui et al., Plant cell Report, 9, pp514-516, 1991). Since then, several relevant studies have been published (Choi and Boe, Korean Journal of Plant Tissue Culture, 20, pp261-266, 1993; Choi et al., Plant Cell Report, 17, pp 84-88, 1997; Choi et al., Ann of Bot. 83, pp309-314, 1999; Han & Choi, Journal of Plant Biotechnology, 30, pp167-172, 2003).

In case of arthropods, relatively high in-differentiation capacity has been studied, and thus, many studies have been carried out on induction of somatic embryos and plant regeneration using callus culture and leaf tissues (Lee and Soh 1993a Korean Journal of Tissue Culture 20: 77-83 1993b) Korean Journal of Tissue Culture 21: 167-171; Park et al 1994a Kor. J Med crop sci 2: 241-245, 1994b J Oriental Bot Res 7: 133-136; Moon et al 1998; Moon et al 1999 Korean Society of Plant Tissue Culture 26: 275-280).

Yin-tree is a genus 1 species that grows only in Northeast Asia, such as Korea, Japan, China, and Eastern Russia, and is classified as a mutant species such as hairy tree and thin-leaf vine. It is in a loss state.

It is known that seed growth, root cutting and grafting are possible for seed growth, but seed breeding is a double dormancy seed with two-year germination characteristics. In the case of thorn trees without seed, it is impossible to breed them.

Conventional methods of breeding barley-free trees have been developed to select varieties of barley varieties and then fix them and grow them through asexual propagation methods such as pedicle grafting and root cuttings. It has to be done in consideration of the timing, and there are difficulties such as much labor and time.

As such, when breeding is difficult or when a lot of time and labor are required for breeding, it is necessary to produce a large number of seedlings using tissue culture technology, which is a practical and effective means of breeding.

The present invention induces somatic embryos using callus cultured aseptically in the aircraft through plant lobe fragments to produce seedlings, and the second germ-derived callus and secondary somatic embryos through the produced seedlings and plants The present invention was completed by producing a large amount of seedlings and in-plant plants by culturing in a solid and liquid medium, and cultivating and transplanting fine seedlings without thorns in a suitable soil.

The prior art related to the present invention, Korean Patent Registration No. 10-0365491, "Animal breeding of new varieties of thorny trees and seed germination promotion method" is the asexual breeding method by pedicle grafting and root cutting of new varieties of vines and plant growth control materials and post-treatment treatment It relates to a method for providing a method for promoting seed germination by.

The method of somatic embryo development and mass production of seedlings from embryonic embryonic cell culture of acupuncture of Korean Patent Registration No. 10-0436632 is obtained by culturing the cotyledons, embryos, roots and young vases of seedlings germinated from seed-derived zygotic embryos. Callus is derived from and when the callus is cultured in a medium to which various kinds of plant growth regulators are added to a method of inducing embryogenic callus and somatic cells.

Korean Patent Registration No. 10-323571, "Preparation of Seedlings from Visceral Galle Embryonic Cells," suspends and cultures the embryonic cells of spiny gallbladder in MS liquid medium to mass-produce seedlings through a somatic embryogenesis process and transfer them to agar medium. It is a method for producing sterile gourd skin in a sterile, pollution-free state.

However, these prior arts have different technical configurations from the present invention.

It is an object of the present invention to induce embryonic callus and somatic embryos formed through in-flight culture and transfer them to the appropriate medium to produce seedlings, and then to induce secondary embryogenic callus and somatic embryos from the produced seedlings. It is to provide a method for producing thorny seedling seedlings in large quantities by producing, growing and purifying.

The present invention has excellent pharmacological effects, excellent taste or aroma, and no thorns for cultivation and management of cultivars, which are growing in demand as high-quality wood and edible or medicinal species, as compared to the conventional vines. It can produce large quantities of thorn-free wood that can increase productivity.

The present invention is a method of producing seedlings in a large amount by producing and purifying seedlings through secondary somatic embryos from thorny foliar disease, somatic embryos by cell culture of thorny vines in the first to seventh steps below. Formation and plant mass production methods can be achieved.

First step; Inducing embryogenic cells and callus from the foliar disease of Kalopanax septemlobus (Thunb. Koidz.),

Second step; Inducing somatic embryos from embryogenic callus,

Third step; Inducing and growing seedlings from induced somatic embryos,

The fourth step; Inducing and propagating secondary embryogenic callus using cultured in-flight plants,

The fifth step; Inducing a large amount of somatic embryos using the induced secondary embryogenic callus,

Sixth step; Mass production of seedlings from secondary somatic embryos,

Seventh step; And purifying the in-flight plants produced in the soil.

Hereinafter, the method of the present invention will be described in detail step by step.

1) inducing embryogenic callus from the lobe tissue (see FIG. 1)

The leaf tissues are collected and sterilized and then placed on callus-inducing medium. The younger the tissues collected for inducing callus, the better the young tissue. The callus induction medium was added with 2,4-DCPA (dichlorophenoxy acetic acid) alone or TDZ; Thidiazuron (1-phenyl-3- (1,2,3- MS (Murashige and Skoog) media with thiadiazol-5-yl) urea) are suitable. Much of the callus produced contains embryogenic callus. Embryonic callus has the characteristics of typical embryogenic callus in pale yellow, low moisture and brittle form.

2) inducing somatic embryos from embryogenic callus (see FIGS. 2 and 3)

Somatic embryos are induced in a medium in which embryogenic callus induced and propagated from leaf tissues does not contain plant growth regulators and the MS salt concentration is reduced to 1/2.

Somatic embryos are embryos derived from plant somatic tissues that can become plants, and develop into seedlings through the stages of globular, heart, and torpedo types.

3) inducing and growing seedlings from induced somatic embryos (see FIGS. 4, 5, 6a, 6b and 6c)

Since the resulting somatic embryos contain a significant number of embryos per callus, the somatic embryos are separated into a certain size and subcultured in a medium in which the MS salt concentration is reduced to 1/2.

Seedlings are derived from somatic embryos in 1/2 MS medium. Induction of seedlings was carried out in the concentration range of 1% to 3% added sucrose.

Good seedlings were induced and grown in a medium containing 1% sucrose at a 1/2 MS salt concentration. Since the seedlings are also derived from several embryos, several seedlings are produced at one time. It is preferable to separate and culture each of the in-plant plants after one month of culture. When seedlings are passaged, it is best to grow two seedlings in a 250 ml culture bottle.

After 8 weeks of isolation culture, each in-flight plant may be one selected from burmeculite, pearlite, peat moss, or may be purified by transplantation to two or more artificial soils to produce seedlings.

4) inducing secondary embryogenic callus from the cultured in-flight plants (see FIG. 7)

Lobe, including some of the roots, leaves, and cotyledon of cultured intestinal plants, can be cut and subjected to MS medium added with 2,4-DCPA alone or with TDZ to induce secondary embryogenic callus.

Each section was cut to a size of about 0.5 to 1 cm and then toothed 4 to 6 sections per Petri dish. Both leaflets, including roots and cotyledons, can induce good embryogenic callus, which is passaged every four weeks in the same medium for proliferation and maintenance. Care should be taken to control callus density and the timing of subculture during subculture.

5) inducing and producing a large amount of somatic embryos using the induced secondary embryogenic callus (see FIGS. 8A, 8B and 9).

It is possible to induce and produce a large amount of somatic embryos in the same way as step 2). However, the second embryogenic callus is easily browned and dies when transplanted into a low salt medium containing no plant growth regulator.

To solve this problem, inducing somatic embryos in medium supplemented with 0.1 to 0.2 mg / L of 2,4-DCPA in MS or 1/2 MS medium can lower browning and mortality of secondary callus, Can be derived.

In the case of a solid medium, somatic embryos derived are present in agglomerates, so it is difficult to separate them. In order to solve this difficulty, it is cultured in a liquid medium having the same composition as that of the solid medium but without the addition of gelite.

In the case of culturing through liquid medium, since each medium is separated by rotation in a rotary stirred incubator, it is not necessary to separate the vessels separately, and it is possible to produce a larger number of times than the solid medium.

The culture of liquid medium should be done once a week to remove about half of the total medium volume before supplying fresh medium.

6) Mass production of seedlings from secondary somatic embryos (see FIG. 10)

Secondary somatic embryos generated in solid and liquid media are separated into appropriate numbers and developed as a seedling when continuously passaged to a medium with low MS salt concentration.

Inducing somatic embryos in medium supplemented with 0.1 to 0.2 mg / L of 2,4-DCPA in MS or 1/2 MS medium and transferring them to 1 / 2MS medium containing no plant growth regulators effectively prevented the seedlings. Can be induced.

Transferring from 1/3 and 1/4 MS medium supplemented with 1% of sugar to grow derived seedlings can prevent overhydration of the plants and effectively grow the plants. In the case of seedlings cultured in a solid medium, each plant must be separated and transplanted into a culture bottle, but in the case of liquid culture, only seedlings of a certain size are transplanted into a solid medium.

For in-flight plants, it is effective to put 30 ml of medium in a 250 ml culture bottle and transplant two plants. In-flight plants transferred to the culture bottle are incubated for about 8 weeks, and subcultured at 4 week intervals shows good results for plant growth. The culture conditions can be illuminated for 24 hours with 24 ± 1 ° C. and 24 μmol m ⁻² s ⁻¹ white fluorescent lamps, preferably for 10 to 24 hours.

7) Purifying the in-flight plants produced in the soil (see Fig. 11)

In-flight plants grow to a size of 10 to 20 cm in 8 weeks of cultivation, allowing the soil to be purified. Take out the in-plant plants of the size that can be purified to soil and wash them with running water to completely remove the medium attached to the plants.

Artificial soil for plant soil purification is used after sterilization using a sterilizer. Sterilized Vermiculrite, Pearlite, Peatmoss, horticulture (Klasmann clay) may be used alone or in combination of two or more. Among them, purified plants in soil mixed with burmeculite, pearlite, and peat moss have the best soil sticking rate.

During the period of hydration, low concentrations of MS salts containing no plant growth regulators and sugars are dissolved in distilled water and sprayed two to three times a week for three weeks. After that, the distilled water is continuously sprayed at the same interval as before until the greenhouse.

Soil purifying of in-flight plants is carried out under light conditions of 10 to 24 hours, preferably 16 hours, at 24 ± 1 ° C and 45% of humidity. Plants cultured for 8 weeks are transplanted into larger pots for transplantation. It grows by varying the degree of shading depending on the climate in a glass greenhouse equipped with a shading film for 2 to 4 weeks. During the period in the greenhouse, watering is enough for one to two hours at intervals of three to four days.

Hereinafter, the content of the present invention will be specifically illustrated by way of examples. However, these embodiments are one embodiment of the present invention, the content and scope of the present invention is not limited to these embodiments.

Example 1 Induction of Callus-Derived Embryonic Cells and Callus without Spiny

The leaves and defoliation of thorny vines are collected, sterilized with 70% ethanol for 45 seconds, then sterilized again with 4% sodium hypochlorite (NaOCl) for 10 minutes and washed with sterilized water at least three times. Sterilized leaf and leaf tissues were added to MS (Murashige and Skoog) medium containing 3% sucrose and IAA, NAA, 2,4-DCPA, BAP, TDZ, 2-iP, Kinetine, GA, and ABA alone or in combination. After culturing for 4 weeks with a 1-1.5 cm long section, callus was formed in a medium containing 2,4-DCPA. In particular, MS with 2,4-DCPA 1 mg / l and TDZ 0.1 mg / l was added. It shows the best callus formation in the medium.

It can be observed that much of the callus produced contains embryogenic callus. Culture conditions for incubation of the dentured tissues are illuminated with 24 ± 1 ° C., 24 μmol m ⁻² s ⁻¹ white fluorescent lamp for 16 hours. The resulting callus can be propagated by continuous transplantation into the same medium. The resulting callus should be passaged at intervals of 3 to 4 weeks and maintained at an appropriate inoculation density. Callus easily browns and dies if the subculture period and proper density are not maintained.

Example 2 Induction of Somatic Embryos from Embryonic Callus

The resulting embryogenic callus was inoculated with medium containing no plant growth regulators, 1.5% sucrose and 0.3% Gelrite, and the MS salt concentration adjusted to pH 5.8 to 1/2. Somatic embryos are induced from embryogenic callus 4-5 weeks after inoculation.

Example 3 Induction of Somatic Embryonic Seedling Plants

After dividing the resulting somatic embryo mass into a certain size and transferring to a solid medium and a liquid medium in which 1% sucrose was added to 1/2 MS medium, cotyledons and roots began to grow after 3 weeks and form a complete seedling plant after 4-6 weeks. Indicates.

Since many seedlings are entangled at once, each seedling is separated one by one and cultured in two 250ml culture bottles. Each seedling is grown in the same medium at one month intervals.

The cultured plants can be used as a material for inducing secondary callus, and can also be produced as thorny seedlings by direct soil purification (the same method as in Example 6 below).

Example 4 Induction of Secondary Embryogenic Callus and Secondary Somatic Embryos through Inflight Seedlings

Cut in-plant plants grown to a certain size (10-15cm) into leaflets containing roots, leaves, and some leaves, and heal them under medium conditions such as 1). Callus starts to form after 2 weeks, and 4cm in diameter after 4 weeks. Is formed.

Callus derived from root tissues as well as some leafy tissues, including some leaves, contain large amounts of embryogenic callus. In order to proliferate and maintain the formed callus, the same medium is passaged every four weeks.

At this time, the interval between each callus should be two 1cm diameter callus in a 250ml culture bottle. Callus rapidly browns and dies if the callus subculture period and interval are not properly maintained. Sections for induction of embryogenic callus show the best results in sections using root tissue.

Proliferated embryogenic callus is transferred to MS or 1/2 MS solids (0.3% Gelrite added) and liquid medium to which 0.1 to 0.2 mg / L of 2,4-D is added, and then a large amount of somatic embryogenesis is induced. In the case of liquid culture, put 50ml of medium and 3g of callus in a 250ml Erlenmeyer flask, and incubate in a rotary stirrer set at a rotational speed of 120rpm and a temperature of 24 ° C.

In the case of liquid culture, remove 25 ml of medium at a weekly interval and add the same amount of fresh medium. In the case of liquid culture, browning and killing are easier than the solid medium if the subculture period and proper density are not maintained.

Example 5 Mass Production of Seedling Plants from Secondary Somatic Embryos

When somatic embryos derived from medium added with 10 g / L of sucrose in 1/2 MS medium were divided into a predetermined size and transferred, the roots and cotyledons began to develop after 3 weeks, and showed a complete in-flight plant shape after 6 weeks.

In-flight plants can be prevented from overhydration of the in-flight plants by effectively reducing the salt concentration of 1% sucrose to 1/3 and 1/4 medium. Each in-flight plant is incubated separately in two 250ml culture bottles. Incubate with 24 ± 1 ° C., 24 μmol m ⁻² s ⁻¹ white fluorescent lamp under 16 hours of illumination and transfer each seedling to the same medium for 8 weeks.

<Example 6> Purification of soil of in-flight plants

After 8 weeks of incubation, the plant is removed from the culture bottle, and the medium is completely removed with running water. The sterilized Vermiculrite, Perlite and Peatmoss are purified in a 1: 1: 1 mixture of soil. do.

During the first two weeks of the purification period, dissolve in distilled water at a concentration of 1/4 MS without addition of plant growth regulators and sucrose and spray spray 800 ml m ^-2 every 3 days, and 1/8 MS for three to six weeks. Spray spray 800 ml m ^-2 once each time. Spray spray the same amount of distilled water after 6 weeks until transfer to greenhouse.

Cultivated plants were cultured under 24 ± 1 ° C, 45% humidity, and 16 hours of light conditions. Purified plants cultured for 8 weeks had Vermiculrite, Pearlite, and Peatmoss, respectively. After transplanting into large pots with 1: 1 mixed soil, grow them by transplanting them into 50% shaded glass greenhouse for 2-4 weeks for soil transplantation. During this period, watering should be done for one to two hours at intervals of three to four days.

The mass production method of the thorny seedling seedlings of the present invention can replace the conventional thorny trees that have been difficult to cultivate and manage the existing, and the mass of thorny seedlings that can solve the problem of the past breeding of rare thorny thorny trees The growth method enables mass production of thorny seedlings throughout the year.

In addition, a small facility can continuously produce large quantities of news items, and can secure stable thorny seedlings.

Claims (15)

Induction of callus in medium containing 2,4-DCPA (dichlorophenoxy acetic acid) and TDZ (1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea) Steps, Inducing somatic embryos using the induced callus, Producing seedlings using the induced somatic embryos, Inducing callus using any one selected from the roots, leaves, or leaf diseases of the produced seedlings, Inducing secondary somatic embryos using the induced secondary callus, Producing secondary seedlings using the induced secondary somatic embryos, Culturing the produced seedlings, A method for mass production of barley-free trees, comprising the step of purifying the induced in-plant plants and adapting the soil-purified plants to shading greenhouses. The medium of claim 1, wherein the medium contains 1 mg / l of 2,4-DCPA (dichlorophenoxy acetic acid) and 0.1 mg / l of TDZ (1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea). And Sucrose 3.0% and Gelrite 0.3%, the mass production method of thorn-free wood, characterized in that the MS (Murashige and Skoog) medium adjusted to pH 5.8. The induced callus was added to 1 mg / l of 2,4-DCPA, 0.1 mg / l of TDZ, 3.0% of Sucrose, and 0.3% of Gelrite, and maintained in MS medium adjusted to pH 5.8. A mass production method of thorn-free wood, characterized by multiplication. The method of claim 1, wherein the induced and propagated callus is grown in a half MS medium adjusted to pH 5.8 by adding 1.5% of Sucrose and 0.3% Gelrite. The method of claim 1, wherein the induced somatic embryos are added to 1.0% Sucrose and 0.3% Gelrite to transfer to 1/2 MS medium adjusted to pH5.8 to induce seedlings Mass production method. According to claim 1, wherein the roots and some leaf cotyledon and leaf tissue of the seedlings produced, 2,4-DCPA 1mg / l, TDZ 0.1mg / l, 3% Sucrose and 0.3% Gelrite Adding, incubating in MS medium adjusted to pH 5.8 to induce secondary callus. The induced callus was maintained in MS medium adjusted to pH 5.8 by adding 1 mg / l 2,4-DCPA, 0.1 mg / l TDZ, 3% Sucrose and 0.3% Gelrite. A mass production method of thorn-free wood, characterized by multiplication. 2. The barb of claim 1, wherein the induced secondary embryogenic callus induces secondary somatic embryos in MS or 1/2 MS medium supplemented with 2,4-DCPA 0.1-0.2 mg / l. Mass production method. The method of claim 1, wherein the tissue containing the induced secondary embryogenic callus and somatic embryos is added 1.5% Sucrose to induce a large amount of secondary somatic embryos and seedlings in 1/2 MS liquid medium adjusted to pH 5.8. A mass production method of thorn-free wood, characterized in that. The method according to claim 1, wherein the induced secondary somatic embryos are added with 1.0% Sucrose and 0.3% Gelrite to transfer to 1/2 MS medium adjusted to pH 5.8 to induce and produce seedlings. Mass production method of thorny wood. The method of claim 1, wherein the seedlings are grown by subcultured with 1/3 and 1/4 MS medium adjusted to pH 5.8 by adding 1.0% Sucrose and 0.3% Gelrite. Mass production method. The method of claim 1, characterized in that the subculture and grown seedlings are incubated for 8 weeks to purify the seedlings using any one selected from burmeulite, pearlite, peat moss, or mixed soil. How to mass-produce thorny trees. The method according to claim 11, wherein during a period of 1 to 2 weeks of purifying period, spray 1/4 ml of plant growth regulator and sucrose free every 800 ml / m ² once every 3 days, until 1/2 weeks. Method of mass production of thorn-free wood, characterized in that spraying 8MS every 3 days 800ml / m ² and then with distilled water. delete delete

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