KR20030030348A - method of organogenesis and somatic embryogenesis for cultivation of single cell - Google Patents

Abstract

PURPOSE: Provided is organogenesis or somatic embryogenesis of ginseng from callus by monocyte culture, thereby massively producing ginseng and allowing clonal propagation of other plants. CONSTITUTION: The organogenesis or somatic embryogenesis of ginseng from callus by monocyte culture comprises the steps of: suspending calluses of ginseng and culturing them; separating monocytes therefrom, followed by nurse culturing them to promote cell division; and progressing organogenesis or somatic embryogenesis of the culture cell by plant growth regulating factors and the control of in vitro condition.

Description

Method for organogenesis and somatic embryogenesis for cultivation of single cell by wild cell culture derived from callus

An object of the present invention is to mass-reproduce wild ginseng known as rare herbs in Korea using cell culture techniques, and in particular, to a method of simply replicating a plant by two processes of organ differentiation and somatic embryo differentiation.

The present invention relates to a plant cloning method by single cell culture derived from wild ginseng callus. Ginseng cloning technology has been reported in many domestic and overseas journals, but in the case of wild ginseng has not yet been systematically researched other than the method of differentiating plants in callus. Although differentiation of plants from callus is generally used, stem differentiation is relatively easy in ginseng and wild ginseng, but stems are differentiated in specific parts of callus even when roots are not developed again after stem differentiation or when regenerated into complete plants. After the differentiation of the roots at different sites, the complete plant is regenerated, and even though the catheter tissue is weakly developed between the stem and the root, it is known that the survival rate during the purification process is lowered.

In the present invention, in order to replicate a large amount of wild ginseng using a single cell culture technology of plants ① a method for separating single cells from callus by a simple method, ② a method for promoting the division of single cells by nursing culture known to be difficult until now , ③ Method to increase organ differentiation rate by chemical treatment in single cell-derived colonies, ④ Regeneration of plants by PEDC (pre-embryogenic determined cell) method by suspension culture of single cell-derived colonies, ⑤ from single cell-derived colonies After the callus was obtained, the method of regenerating the plants by the induced embryogenic determined cell (IEDC) method and the ⑥ bioreactor to develop a simple and efficient process to increase the survival rate and speed the growth of the differentiated plants.

1 is a cell culture derived from wild ginseng callus,

2 is a single cell isolated from the liquid culture,

3 is a photograph nursing cultured single cells,

4 is a state in which cells are divided from single cells to form colonies,

5 shows somatic embryonic cell mass derived from a single cell,

6 is a photograph showing stem subdivision from somatic embryonic cell mass,

Figure 7 shows the state of the somatic embryo,

Figure 8 is a photograph showing a wild ginseng plant completely reproduced.

In the present invention, the callus of wild ginseng was cultured in liquid suspension at high speed, so that the cultured cells were separated from each other and then single cells were separated therefrom and used as a material of the invention. The isolated single cell itself is difficult to divide the culture medium using nutrients effectively nursing it to obtain a divided cell aggregates colonies. The cell colonies were able to differentiate themselves by treating plant growth regulators in the culture medium, noting that they were capable of differentiating themselves. In order to obtain genetically safe plants rather than organ differentiation, two somatic embryos were induced. The first was to divide colonies into sufficient numbers using liquid suspension culture technology, and then induced somatic embryos by PEDC method. As a plant obtained by the IEDC method using a callus obtained by culturing the colonies for about 4 more weeks.

[Test Example 1]

The method for mass reproduction of complete plants through organ and somatic embryo differentiation by single cell culture of wild ginseng is as follows. Wild ginseng callus was used after subculture at least six times in a new culture medium to improve cell division capacity before use as a sample. Known plant tissue culture media can be used for this purpose, for example mB5 (modified Gamborg's B5), Durzan, MS (Murashige and Skoog), DKW (Driver-Kuniyuk-Walnut), GD (Gresshoff and Doy), SH (Schenk and Hildebrandt), LP (Quoirin and Lepiovre), and WPM (woody plant medium) medium.

Both of these media can be used, and various additives can be added or some of the components can be removed if necessary. Particularly preferred among them is WPM medium. In this way, the same or different media as those described above may be used for liquid suspension culture from callus in the logarithmic growth period, but it is advantageous to use WPM and MS media in terms of growth of cultured cells.

When disinfecting at 121 ℃ for 30 minutes without adding agar, it was prepared as a liquid culture medium, and then sterilized by adding 5 g of sterile chopped callus biomass and 200 ml of prepared liquid medium to a sterilized 500 ml Erlenmeyer flask. After sealing with aluminum foil, the mixture was stirred at a speed of about 110 rpm in a stirrer. In the culture medium, oxine was added as a plant growth regulator. Especially, when 2,4-D was added at 1 to 5 ppm, not only cell growth but also attenuation of cell cohesion was increased to increase the frequency of single cell and several cell aggregates. . To cultivate single cells from the liquid suspension culture, the cell populations that were passed through a mesh of 60 mash were firstly selected. In order to separate the single cells, 2 g of cellulase was added to the culture medium so that the final concentration was 1%. After stirring at low speed of 10 to 30 rpm for about 20 minutes at 26 ℃, washed three times with fresh liquid culture medium. Secondly, for single cell separation, 10 gradient solutions were prepared from 1% to 10% concentrations of sugar in culture medium, and then sterilized and left at 4 ° C for 24 hours. Ten layers were formed by dispensing 3 ml of the concentration gradient solution into the test tubes in the order of high to low concentration.

The cell cultures collected by cellulase treatment are mixed well with 10 ml of culture medium, gently poured into the prepared test tube wall, and left for about 5 minutes to separate the cell population according to the weight difference. Observing the cell group formed at the top under a microscope, the cells of the layer containing relatively only single cells are selectively separated and rinsed two to three times with a new culture medium. The prepared single cells can be cultured by the previously reported liquid plating, liquid drop, and hanging drop methods, but the nursing culture method was most effective to increase colony formation rate. For nursing culture, cut the upper part of the callus, which has been incubated for about 1 week in solid medium, sterilize it about 2 mm, sterilize it on the liquid culture medium, and place the filter paper (Whatman No. 2) where water is absorbed. Single cells were cultured under the microscope on the filter paper, and when cultivating a large amount, the culture medium was absorbed by the filter paper by gently raising the single cells at a concentration of about 1 102 per ml on dry filter paper after sterilization, and the single cells were filtered. To remain on the stomach. Colony formation was observed after 4 to 6 weeks of culture in single cell nursing cultures. In order to cause organ differentiation from colonies, a medium in which a cytokine line of plant growth regulators were added in a range of 0.1 to 10 ppm was used in a solid medium made by hardening about 0.75% of agar in the aforementioned culture medium. As cytokinin, BA, 2iP, kinetin, and zeatin can be used, but considering the cost and efficiency, it was better to add BA to 0.2 to 5 ppm.

Inoculation of the colonies into the organ differentiation medium caused necrosis of about 3/4 but the other colonies turned green to form stems. Root development was observed when stem-derived colony mass was transplanted into a culture medium containing no plant growth regulators or containing about 1 to 5 ppm of NAA or in a medium containing 0.001 to 0.01 ppm AgNO3 in the same medium. . When colonies were incubated in MS or WPM medium containing 2 to 4-ppm of 2,4-D, the callus surface induced irregular yellow callus and transparent, white or pale yellow callus with slightly rounded surface. . When the rounded callus was transplanted to MS or WPM medium containing no plant growth regulator, IEDC-type somatic embryos were observed within 4 weeks. At this time, more than about 25 somatic embryos were formed per callus of about 0.5 cm in diameter, and when they were passaged to the same medium one by one, about 60% of them were differentiated into complete plants. When the callus obtained by several passages of somatic embryos of IEDC type was cultured in the same manner as described above, numerous somatic embryonic cells were obtained, and the cells floating in the upper layer of the medium during culture were not differentiated. Removed at incubation. The growth rate of somatic embryonic cells was different, and when cultured for about 4 weeks, somatic embryos of round, heart and torpedo types were observed. At this time, after separating the round somatic embryos, most somatic embryos developed as complete plants when transplanted into semi-solid MS or WPM medium containing about 0.5% of agar. Plants obtained through organ differentiation and somatic embryo formation by two methods were able to grow about 75% in solid culture medium containing 0.01% activated charcoal, but they were maintained at low brightness due to necrosis of leaves when light over 100 lux was given. It was effective. For the rapid growth of wild ginseng, the growth was excellent in the air-rich bioreactor or the Ebb and flood bioreactor. The air floatation bioreactor was made with a conical flask or a round flask, with an air outlet at the top and a fine glass filter at the bottom, and a micro filter with 0.2 micrometre holes at the air outlet and the glass filter. . The size of the incubator was 1 liter, and the height of the culture solution was adjusted so that about 1/3 of the stems were submerged when the wild tricuspids were cultured. At this time, the flow of external air was maintained to 0.01 vvm per minute. The Ebb and flood bioreactor mainly used a modified round flask, and a 0.3-diameter square perforated net was used inside the incubator. The culture medium was kept in the bioreactor for 1 hour and stored for 3 hours. The culture medium was collected in a container, and the control was performed continuously using a timer and a solenoid valve. Bioreactor culture was also carried out in aseptic way.

[Test Example 2]

The plant reproduction method by single cell culture completed in the present invention has already been demonstrated in the Poplar tree, and the plant reproduction by the nursing culture of single cells is stable in some species by obtaining the same result in ginseng, prickly pear and arbor. It has been found that it can be used for nutrition propagation.

The characteristics and effects of the present invention on the organ and somatic embryo differentiation method of wild ginseng by single cell culture are as follows. Plant differentiation by single cell culture has been evaluated as a breakthrough in breeding research using plant somatic mutations, but until now, there have been reports of successful mass replication only in several species, such as tobacco and carrot, and the first in ginseng and wild ginseng has been reported. By succeeding, I think it will be able to provide a new paradigm for Korean ginseng breeding. Mass reproduction is possible within a short time by replicating plants from colonies derived from single cells. In the case of plant organ differentiation using ginseng and wild ginseng callus, there is a report that the survival rate is greatly reduced during the purification process. It was about 75%. Until now, plant cloning was mostly dependent on organ differentiation or IEDC method, but by developing the cloning technology by PEDC method, it is useful not only for mass replication of wild ginseng, but also when applying the technology shown in the present invention to other species. Nutritional breeding is likely to be possible.

Claims (6)

Differentiation of wild ginseng's organs and somatic embryos by callus-derived single cell culture, wherein the callus of wild ginseng releases cells using an enzyme such as cellulose, and then separates the free cell population using only a single cell using a sugar gradient Way. The method of claim 1, A method for differentiating organs and somatic embryos of wild ginseng by callus-derived single cell culture, which promotes cell division of single cells by nursing culture and induces colonies. The method of claim 2, Conduit of stems and roots formed from colonies by using a medium containing a plant growth regulator or a medium containing AgNO3 as a method for differentiating to a complete plant through organ differentiation and colonization formed by the nursing culture A method for differentiating organs and somatic embryos of wild ginseng by single cell culture derived from callus, wherein the tissues are connected as one. The method of claim 2, Method for differentiating organs and somatic embryos of wild ginseng by callus-derived single cell culture, characterized in that differentiation of somatic embryos directly in the form of IEDC in the colony. The method of claim 2, The wild ginseng by callus-derived single cell culture, characterized in that the colonies are grown in callus, to establish a suspension culture, and to extract only the round somatic embryos from the suspended somatic embryos to differentiate somatic embryos in semi-solid medium. Organ and somatic embryo differentiation method. A method for differentiating organs and somatic embryos of wild ginseng by single cell culture derived from callus, characterized in that the plant obtained through organ differentiation and somatic embryo differentiation is purified using a bioreactor.