KR100569507B1 - The culturing apparatus for periodically submerged culture and the culturing method thereof in the somatic embryos culture for mass production of Eleutherococcus senticosus seedlings - Google Patents

Abstract

The present invention relates to a cyclic immersion culture vessel and a culture method used for culturing somatic embryos for mass production of Eleutherococcus senticosus seedlings, and specifically, a medium storage by freely controlling the air injection using a solenoid valve and a timer. The present invention relates to a periodic immersion culture vessel capable of periodically immersing the nutrient medium in the culture vessel in which the charging culture unit is separated, and a culture method using the same.

Periodic immersion culture vessel and the culture method using the same of the present invention can increase the conversion rate to seedlings and shorten the conversion period by inducing nutrient supply and water stress reduction to the somatic cell culture, conventional agar culture method or suspension culture method Compared to the production of high quality seedlings, it can be useful because the production cost can be reduced by the introduction of automation.

Periodic immersion culture, somatic cell culture, prick

Description

The culturing apparatus for periodically submerged culture and the culturing method approximately in the somatic embryos culture for mass production of Eleutherococcus senticosus seedlings}             

Figure 1a is a design of the culture vessel for the periodic immersion culture of the present invention,

Figure 1b is a schematic diagram showing the operating principle of the periodic immersion culture vessel,

Figure 2 is a diagram showing the structure of the purified vessel that can be purified from the seedlings converted from the prickly gallium somatic embryo cultured using the culture vessel of the present invention,

Figure 3a is a photograph of a small bioreactor for mass propagation of somatic embryos from embryonic cells prior to cyclic submerged culture of prickly somatic embryos,

Figure 3b is a photograph of the actual culture vessel that is subjected to the periodic immersion culture method,

Figure 3c is a photograph of a state of immersing the medium by air injection in the periodic immersion culture method,

3D is a photograph of a state in which the medium is being dried by blocking the air injection in the periodic immersion culture method,

Figure 4a is a photograph showing that malformed somatic embryos in the bioreactor culture method,

Figure 4b is a photograph showing the mass production of the individual in the normal cotyledon state in the periodic immersion culture method,

Figure 5a is a photograph of the purified container of the present invention,

Figure 5b is a photograph of the thorn seedlings gourds purified after two weeks in the purified container of the present invention,

Figure 5c is a photograph of a cultured prickly thorn seedlings to complete the soil purification by replanting the purified seedlings into the soil.

* Explanation of symbols for main parts of the drawings

10: compressor 11: solenoid valve

12: air conditioner 13: air outlet

14: culture unit 15: medium storage unit

16: Glass filter 17: Medium transfer passage

18: Nozzle for medium exchange 19: Timer

20: body 21: lid

22: air injection device 23: plastic rod

24: plastic mesh

The present invention relates to a periodic immersion culture vessel and a culture method used for culturing somatic embryos for mass production of Eleutherococcus senticosus seedlings, and more specifically to free air injection using a solenoid valve and a timer. The present invention relates to a periodic immersion culture vessel capable of periodically immersing a nutrient medium in a culture vessel in which a medium storage unit and a culture unit are separated, and a culture method using the same.

Prickly Pear is a tree plant belonging to the family Arboraceae, and it is known to grow natively in southeastern Siberia, Japan's Hokkaido, parts of China, and northern part of Gangwon-do and Deogyusan area. Prickly Pear is used for medicinal roots, leaves, or bark, and is known to be particularly effective in improving energy, diabetes, anticancer, anti-inflammatory, neuralgia or life extension (Yook et al ., Kor. J. Pharmacog 7: 179-190, 1976). Germination of prickly pear seeds requires over three years of ripening in nature and can be artificially layered over 18 months (Kuribayashi and Ohashi, Soyakugaku Zasshi 25: 95-101 , 1971; Isoda and Shoji, Natural Medicine 48: 75-81, 1994), which is difficult to germinate even with the above treatment, making it difficult to breed in large quantities. As such, for plants that are difficult to reproduce, it may be a significant means to produce seedlings in large quantities using tissue culture techniques in a laboratory.

Recently, there is a breeding method using somatic embryos, which is a popular way of breeding prickly pears, which is an economical breeding method that exhibits high productivity at low cost without problems such as pesticide contamination or environmental damage that may occur when seed is grown. Gui et al ., Plant Cell Rep . 9: 514-516, 1991, were first reported by Gui et al . (Choi et al ., Annals of Botany 83: 309-314). , 1999; Plant Cell Rep . 20: 1112-1116, 2002). However, the agar culture method used in the culture of somatic embryos of prickly gallium requires a lot of labor due to frequent exchange of agar medium in the process of producing seedlings, and the suspension culture method is converted in the process of converting somatic embryos into seedlings. Low efficiency has many problems to use practically.

Therefore, the present inventors periodically nourish medium in the culture vessel by using a culture vessel in which the solenoid valve and the timer are attached and the medium reservoir and the culture unit are separated in the somatic cell culture for mass production of Eleutherococcus senticosus seedlings. A method of immersion has been developed. As a result of culturing the soybean embryos in accordance with the periodic immersion culture vessel of the present invention and the culturing method using the same, it was confirmed that the production of sapling plants of high quality thorns can be completed and completed the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a periodic immersion culture container capable of producing a large amount of individuals having excellent conversion rate from somatic embryos to seedlings in mass production of prickly seedling seedlings using somatic embryos and a culture method using the same.

In order to achieve the above object, the present invention provides a culture vessel equipped with a solenoid valve and a timer, the storage unit and the culture unit of the medium are separated, and a method for culturing the somatic embryos using the same.

In addition, the present invention provides a mass production method of thorn seedlings using the culture vessel and the culture method.

Hereinafter, the present invention will be described in detail with reference to FIGS . 1A to 3D .

The present invention provides a culture vessel equipped with a solenoid valve and a timer, and the storage portion and the culture portion of the medium are separated.

The culture vessel of the present invention is connected to the air regulator 12, the solenoid valve 11, the compressor 10 from the body, the timer 19 is connected to the solenoid valve. The stopper of the body has an air outlet 13 equipped with a sterile filter, and the filter 16 is mounted in the middle portion of the cylindrical body to separate the culture medium and the medium in a dry state and to facilitate the rapid discharge of the medium. . At the bottom of the culture unit 14, there is a medium moving passage 17, which serves to move the medium to the culture unit 14 and the storage unit 15. The medium exchange nozzle 18 is connected to the lower end of the reservoir so that the medium can be easily exchanged (see FIGS . 1A and 1B ).

The material of the culture vessel and the filter 16 of the culture vessel is preferably made of glass or polypropylene, and the thickness of the filter is preferably 1 to 15 mm, but more preferably 3 to 8 mm. . In addition, the pore of the filter is preferably 3 to 50 ㎛, more preferably 10 to 20 ㎛.

Since the air that is always introduced through the air conditioner is not injected into the culture vessel while the solenoid valve is open and is discharged by the solenoid, the culture medium does not reach the glass filter in which the medium is stored. It is in a dry state (see FIG. 3D ). In the state where the solenoid valve is closed, the air introduced through the air regulator is not discharged to the outside, but is introduced into the culture vessel, and the introduced air reaches the medium in the reservoir to the part where the culture is located and is converted into the immersion state ( See FIG. 3C ). The opening and closing of the solenoid responsible for switching between the dry state and the immersion state is controlled by a 24-hour timer.

After culturing the somatic embryos using the culture vessel of the present invention and looking at the differentiation aspect, the somatic embryo produced by the cyclic immersion culture method using the culture vessel of the present invention in the number of formation of total cell embryos, Not only was it superior to other cultures, but the differentiation of seedlings was also rapid. Specifically, in the development stage of somatic embryos differentiated in the order of globular, heart, torpedo, tocotyledon, the initial stage using the culture vessel and the method of the present invention As a result of culturing somatic embryos, the heart stage somatic embryos were increased by 7 times compared to the liquid suspension culture method after 2 weeks of culture.The cotyledon stage somatic embryos were also 1.5 to 2 times higher than other culture methods after 5 weeks of culture. It increased by about.

In addition, the culture vessel of the present invention and the culturing method using the same can reduce the water stress of the culture because it allows periodic immersion of the culture, lower the contamination of the medium, shorten the production process because the medium exchange is simpler than the conventional culture method Labor costs can be reduced.

The present invention provides a method for mass production of prickly seedlings using the periodic immersion culture vessel and the periodic immersion culture method.

The mass production method of the prickly seedling seedling of the present invention includes the steps of: i) inducing embryonic cells from the seedlings germinated from the sterilized prickly seed; Ii) differentiating the embryogenic cells induced in step iii) into somatic embryos; Iii) culturing the somatic embryo obtained in step ii) using the periodic immersion culture vessel of the present invention; Iii) converting the somatic embryo cultured in step iv) to a seedling plant; And, iii) purifying the seedling obtained in step iii).

Periodic immersion culture vessel of the present invention and the cyclic immersion culture method using the same can be applied to the mass production of plant seedlings using tissue culture technology, it is preferable to apply to the mass production of plant seedlings using somatic cell culture, Medicinal plants such as thorny oak tree, ogalpi tree, ginseng, wild ginseng, cheonma, bellflower, peony, peony, industrial wood plants such as pine, cedar, eucalyptus, and flowering plants such as lilies, lilies, calla, daylily, gerbera and statis And it is more preferable to apply to the seedling production of vegetable crops, such as garlic, potatoes, carrots.

In the above-mentioned mass production method of pruning seedlings of the present invention, the following purifying container may be used in the step of purifying the seedlings of step iii).

Purified container provided by the present invention is a device that can control and maintain the humidity to be suitable for the purification of the culture plants.

Specifically, the purified container of the present invention is composed of a body 20 and a lid 21, an air injection device 22, a plastic rod 23, a plastic net 24 and a connecting part (see Fig. 2 ). The air injector 22 on the outside of the body is connected to the plastic rod 23 with holes at regular intervals mounted on the lower end of the body. There is a plastic net 24 to put the culture plant on the plastic rod, filling the water to the degree that the plastic rod is submerged and injecting air from the air injecting device, while creating a moist bubble from the hole of the plastic rod purifying vessel You can maintain the humidity. In addition, since the purified container of the present invention uses a standardized box-shaped body, not only increases the loadability and ease of operation, but also is made of a transparent material to smoothly supply light to the cultured plant and prevent contamination such as mold.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Culture of Prickly Somatic Embryos Using Periodic Dipping Culture Vessel

<1-1> Induction of Embryonic Cells from Seed-derived Cotyledon

Prickly seed was stratified at a low temperature of 10 ° C. for about 2 months and then sterilized for 1 minute with 70% ethanol and 1% sodium hypochlorite solution for 30 minutes. The seed sterilized by the above method was washed three times with sterile water, and the tissue containing the pear was cut to about 1 to 2 mm, and then the cut tissue was treated with 3% sugar and 0.25% gelite. Incubated for two months in the MS (Murashige and Skoog) agar medium. 2 mm of cotyledons and hypocotyls of the seedlings germinated in the culture step were cut and transplanted into MS agar medium to which 0.5 to 1.0 mg / L of 2,4-D was added. Embryonic cells were induced by culturing under conditions ( FIG. 3 ). Embryonic cells induced in the culturing step were maintained by subculture every two weeks in the same medium as the medium composition. At this time, the pH of the agar medium was adjusted to 5.8, and sterilized for 15 minutes at 1 atm at 121 ℃.

<1-2> Mass Growth of Somatic Embryos from Embryogenic Cells

0.5 g of live oocytes embryonic cells obtained in Example <1-1> were cultured in 3 L MS liquid medium containing 1 mg / L 2,4-D and 3% sugar in a 5 L bioreactor. The cells were suspended and cultured at 25 ° C. for 24 hours in dark conditions for 2 weeks. Proliferated embryonic cells were cultured in a medium without 2,4-D for 18 months at 25 ° C. under light conditions of 18 hours in bright and 6 hours in dark and globular and early heart. Somatic embryos were obtained.

<1-3> Cultivation of Somatic Embryos of Prickly Pear Using Periodic Dipping Culture Vessel

In order to examine the effect of the culturing method using the periodic immersion culture vessel of the present invention, the soybean embryos of globula state and initial heart state obtained in Example <1-2> were subjected to various conditions under the conditions described in Table 1 below . The culture method was used to compare the results. As a result of culturing while maintaining the light amount of 20 umolm ^-2 s ^-1 at 25 ℃ under a light cycle of 18 hours, as shown in Table 2 and Figure 4 , the periodic immersion culture vessel of the present invention and a culture method using the same Compared to other culture methods, was not only better in total somatic embryogenesis, but also faster in seedlings. That is, two weeks after the culture, the somatic cell culture at the heart stage was increased by about 7 times compared to the liquid suspension culture method, and after 5 weeks of culture, the somatic embryo at the cotiledon stage was also increased about 1.5 to 2 times compared to other culture methods. On the other hand, in the liquid suspension culture with the highest number of somatic embryo formation per 100 mg, it can be seen that the development of somatic embryos is late because a large number of somatic embryos exist in the early stages of differentiation per unit mg. In addition, the bioreactor culture method of the culture method malformed most of the somatic embryos in the differentiation stage due to excessive air injection ( Fig. 4a ). That is, 5 weeks after culture, the somatic embryo produced in the bioreactor reached about 40% or more, whereas the somatic embryo produced by the culture method using the periodic immersion culture vessel of the present invention had a malformation rate of less than 10%. Indicated. Therefore, it was confirmed that the periodic immersion culture vessel of the present invention and the periodic immersion culture method using the same can be effectively used to mass-produce high-quality thorny oocytes somatic embryos ( FIG. 4B ). Normal somatic embryos that have been differentiated by the periodic immersion culture are transferred to a medium containing 1/2 MS salts (1/2 MS salts, 15 g / L sugar and 50 mg / L myo-inositol) and cultured for 1 to 2 months. It was converted to a seedling.

Example 2 Purification of Prickly Pear Seedling Plant Using Purifying Container

In order to purify the seedlings obtained in Example <1-3>, the purified container of the present invention that can maintain the humidity by injecting air into the container containing distilled water to generate a wet bubble ( Fig. 5a ). Specifically, the seedling seedlings obtained in Example <1-3> were transplanted into a plastic plug seedling containing pit moss and pearlite 1: 3, sufficiently watered, and purified in the purified container for 2 weeks. ( FIG. 5B ). As a result, the survival rate of the seedling seedlings was found to be 90% or more, and the surviving purified seedlings were transplanted into an easy-to-ship port to complete the present invention ( FIG. 5C ).

Periodic immersion culture vessel of the present invention and the cyclic immersion culture method using the same in the mass production of prickly seedling seedlings using somatic embryos, compared to the conventional agar culture or suspension culture method, it is appropriate to reduce the nutrient supply and moisture stress to the cultured somatic embryos It is possible to produce high quality seedlings by inducing and increase the conversion rate to seedlings. In addition, it can be useful because it can achieve a reduction in the conversion period and the labor cost reduction by the automation of the production process.

Claims (10)

The air regulator 12, the solenoid valve 11, and the compressor 10 are connected from the container body, and the timer 19 is connected to the solenoid valve, and the culture unit 14 and the storage unit 15 of the body are connected. Is separated and the stopper of the body has an air outlet 13 equipped with a sterile filter, the middle portion of the culture unit 14 is equipped with a filter 16 and the culture medium passage path to the storage unit at the bottom of the culture unit 14 There is (17) and the storage unit 15, the culture vessel, characterized in that the nozzle for exchange medium 18 is connected. The culture vessel of claim 1, wherein a material of the body and the filter of the culture vessel is glass or polypropylene. The culture vessel of claim 2, wherein the filter has a thickness of 3 to 8 mm and the pore of the filter is 10 to 20 μm. Periodic immersion culture method of thorn ogalpi somatic embryo using the culture vessel of claim 1. Iii) inducing embryonic cells from seedlings germinated from sterilized prickly gill seed; Ii) differentiating the embryogenic cells induced in step iii) into somatic embryos; Iii) culturing the somatic embryo obtained in step ii) using the culture vessel of claim 1; Iii) converting the somatic embryo cultured in step iv) to a seedling plant; And, Iii) a method for mass production of barley seedlings comprising the step of purifying the seedlings obtained in step iii). The method according to claim 5, wherein the purification of the seedlings in the step iii) comprises a transparent body 20 and a lid 21, and a hole at a predetermined interval in which an air injector 22 outside the body is mounted at the lower end of the body. Is connected to the plastic rods 23, the mass production method of thorn seedlings, characterized in that using a purified container equipped with a plastic net 24 to put the culture plant on the plastic rods. A mass production method of plant seedlings using the culture vessel of claim 1. 8. The method of mass production of plant seedlings according to claim 7, wherein the plant is a plant capable of culturing somatic embryos. The method of claim 8, wherein the somatic embryo culture plants are thorny oak tree, oak tree, ginseng, wild ginseng, cheonma, bellflower, peony, peony, pine, cedar, eucalyptus, lily, lily, collar, daylily, gerbera, A mass production method of plant seedlings, characterized in that any one selected from the group consisting of statis, garlic, potatoes and carrots. delete

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