KR20000056501A - Novel process for mass production of embryogenic cells and seedling thereof on hormone independent medium in ginseng - Google Patents

Abstract

PURPOSE: A mass production method of microplant and somatic cell ginseng by suspension culture is provided. CONSTITUTION: A zygote embryo is taken under sterile condition and cut cotyledon is incubated on Murashige and Skoog's medium containing doubled ammonium nitrate, 3% of sucrose and 0.8% of agar at pH 5.8. Embryonic callus is formed on the base of cotyledon after 2 months incubation. 10 successive culture at every 5 weeks in Murashige and Skoog's medium containing doubled ammonium nitrate produces homogeneous cell line. Somatic cell of ginseng is obtained by incubation of callus on one third Murashige and Skoog's medium without any growth regulator. Somatic cell embryo is obtained by incubation on Murashige and Skoog's medium containing 3% of sucrose and 0.8% of agar without any growth regulator. Microplant is obtained by incubation on one third Murashige and Skoog's medium containing 3% of sucrose and 0.8% of agar with germinated somatic cell embryo treated cold at 0°C for 50 days.

Description

NOVEL PROCESS FOR MASS PRODUCTION OF EMBRYOGENIC CELLS AND SEEDLING THEREOF ON HORMONE INDEPENDENT MEDIUM IN GINSENG}

The present invention relates to the induction of embryonic callus from the ginseng embryo, the mass production of embryogenic cells, and the production of somatic embryos and seedlings, especially in medium in which no growth hormone is harmful to humans from cotyledons of ginseng. It is a method of inducing forming callus and producing ginseng cells in large quantities through solid culture and suspension culture, inducing somatic cells from embryonic callus and propagating somatic embryos to produce seedlings.

Ginseng (Panax ginseng C.A. Meyer) is an arboraceae that needs to be sundown, and it is difficult to cultivate due to severe damages such as serial disorders and root diseases. Also, in order to use ginseng root for medicinal purposes, it is necessary to cultivate for about 4 years or more, so many pesticides are damaged and are very expensive. In general, 4 years after sowing one ginseng seed, its weight is about 50g. Also, wild ginseng, also known as elixir, is hardly found in nature due to the difficulty of growing conditions. Meanwhile, there is a method of mass-producing ginseng cells on board using biotechnological methods. When ginseng cells are cultured using the cell culture method, the growth rate of cell masses is much higher than that of the ginseng plants obtained in the field. Ginseng cell culture attempts have been carried out by many researchers (Furuya et al., 1973, Chem. Ang et al., 1989, Proc. 2nd Internal Sym. On recent Advance in Natural Production Research.p 431-449 , Choi et al., 1900, Korean J. Ginseng Sci. 14: 107-111) It has been reported that the saponin content of cultured cells does not drop significantly compared to the ginseng root (Asaka et al., 1993, Plant Med. 59). : 345-346,; Asaka et al., 1993, Lett. 15: 1259-1264). Most of these studies, however, added exogenous growth regulators for the growth of cultured cells. Since 2,4-D, a mainly used growth regulator, is marketed by the manufacturer as carcinogenic, pure purification of the active ingredient was necessary to use the cultured cells grown in 2,4-D. Pure purification of the active ingredient has not been practical because it greatly reduces the efficiency of production costs. If ginseng cultured cells can be grown in basic salts and sugar solutions without the addition of growth regulators that are harmful to the human body, it can be the ideal culture system. However, the method of inducing ginseng cultured cells without the addition of growth regulators has not been successful until now, but Japanese researchers have studied the induction of ginseng cultured cells in medium without growth regulators by high temperature treatment. , Plant Med. 59: 345-346), and recently, a Japanese company called Ildong Major developed various processed foods such as "extract granules by cell culture."

The present invention induces the embryogenic callus from cotyledons of ginseng without changing the concentration of ammoniumate in the incubator without the growth regulator added to ginseng by suspension culture or solid culture without addition of the growth regulator. Cells were produced in large quantities, somatic embryos were derived from embryonic callus derived from ginseng cotyledons and propagated, and seedlings were produced from these somatic cells.

It is an object of the present invention to provide a method for producing ginseng cells in large quantities in a growth hormone-free medium using biotechnological cell culture technology, for the formation of somatic embryos and for the mass production of seedlings.

The purpose of the present invention is to induce embryogenic callus in a medium without growth regulator from cotyledons of ginseng, and to multiply the embryogenic callus in a solid medium and a liquid medium without growth regulator, and from ginseng cotyledons It is achieved by the method of the present invention which produces somatic embryos from induced embryogenic callus followed by seedlings.

1 is a photograph inducing embryogenic callus from the cotyledons of ginseng

Figure 2 is a photograph of inducing somatic embryos from embryogenic callus

Figure 3 is a photograph of a cell line obtained by subcultured for 10 months by selecting a cell mass having uniform cellular characteristics from the embryogenic callus

Figure 4 is a photo of ginseng cells grown by suspension cultured embryogenic cell line

Figure 5 is a photograph of a seedling plant re-differentiated from somatic embryos

Figure 6 is a proliferation effect of ginseng embryogenic callus with different culture concentrations of murascige and squeegee

The present invention induces embryogenic callus from cotyledons of ginseng by changing only the concentration of ammonium in the medium containing no growth regulators and induced embryogenic callus in solid medium and liquid medium containing no growth regulator to ginseng cells. It produces a large amount, and induces somatic embryos from embryogenic callus formed from the ginseng cotyledons and mass production of ginseng seedlings from somatic embryos.

The present invention also provides a method of producing ginseng cells in a large amount by repeating passage of the embryogenic callus obtained as described above to a solid medium and a liquid medium containing no growth regulators and inducing embryogenic callus from the induced somatic embryos. It also relates to a method of proliferating somatic embryos in a large amount by repeating the method.

A feature of the present invention is to induce embryogenic callus in the medium without the growth regulator, and to produce the ginseng cells in a large amount in the medium without the growth regulator, inducing somatic embryos and producing the seedlings.

In the method of the present invention, in order to produce a large amount of ginseng cells and somatic embryos, it is preferable to repeat the method of subculture again using a part of mass-proliferated ginseng cells and a part of the somatic embryos.

Therefore, the present invention is a growth regulator to induce embryogenic callus from the cotyledons of ginseng in the medium without addition, propagating the induced embryogenic callus in a solid medium without the addition of a growth regulator, the growth regulator is induced Suspension culture in liquid medium not added, producing somatic embryos from embryonic callus, inducing embryogenic callus from somatic embryos, seed-derived somatic embryos in seedlings in solid medium without growth regulator It is also related to the method of germination and mass production.

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

1) inducing embryogenic callus from ginseng cotyledon in medium without growth regulator

After aseptically treating mature ginseng seeds, the seeds are dissected to aseptically shed the zygote and then the cotyledons are cut. Cut cotyledons are adjusted to pH 5.8, and no growth regulator is added, and the concentration of ammonium nitrate is doubled in the basal medium of Murashige and Squeeg. All media are incubated in Murashige and Squeeze medium containing 3% sugar and 0.8% agar. At this time, the culture room conditions are illuminated for 24 hours by 24 ± 2 ℃, 24μMm ^-2 s-1 white fluorescent lamp. Embryonic callus is induced at the base of cotyledons after about 2 months in the squeegee medium in which the ammonium nitrate is added twice to the basal medium of Murashige and Squeeg (see Fig. 1). Early embryogenic callus was a mixture of fragile callus and hard callus, but it was passaged 10 times at intervals of 5 weeks in Squeeg's medium, which added twice the ammonium nitrate to the base medium of Murashige and Squeeg, to obtain a uniform cell line. . In the basal medium of murascige and squeegee, somatic embryos are also directly generated at the base of cotyledon. If necessary, the somatic embryo can be propagated to obtain a seedling plant. On the other hand, when ginseng cultivation is cultured in a medium having increased ammonium nitrate without cutting cotyledon in ginseng zygote, callus is formed on the cotyledon surface. If you want, you can use this callus for the next step.

2) culturing the induced embryogenic callus in a solid medium to which no bioregulatory agent is added

To investigate the culture concentration of murascige and squeegus on the growth of embryogenic callus, the cultured concentration of murascige and squeegee was 1 / 3-fold, and the murascige and squeegee were not added to the growth regulator without increasing ammonium nitrate. Subculture 1 g of embryogenic callus into solid and liquid medium containing 10 mL of medium. This cell line is browned and unable to grow due to the secretion of browning material from embryogenic callus from about 1 week of culture in the basal medium of murasigge and scog. However, the growth of the cell lines was maintained rapidly in the medium in which the murasigge and the squag basal medium were lowered to one third the concentration (see FIG. 3). Repeat subculture to produce as many ginseng cell lines as desired. The proliferation of cells does not change significantly depending on the concentration of sugar.

3) suspending the induced embryogenic callus in a liquid medium not added with the life control agent

In a 300 mL flask, add 100 mL of the liquid medium containing no growth regulator with the concentration of Murashige and Squeege to 1/3, and the sugar concentration to 1%, and transfer 500 mg of the embryogenic callus obtained in 2). A large amount of ginseng cell mass is produced by culturing at 100 rotations / minute with a rotary shaker under a light of 500 lux or less. By repeatedly subcultured the obtained embryonic cell mass in the same manner, a large amount of the embryonic cell mass can be obtained as desired (see FIG. 4). In particular, the cell lines were grown in more than 20 liters of bioreactors, allowing for large tank cultures.

4) inducing somatic embryos from embryogenic callus induced in cotyledon

Embryonic callus obtained from the ginseng cotyledon was immediately transferred to a solid medium containing 3% sugar and 0.8% agar, but without the growth regulator, in a normal culture medium and in a culture medium with pH lowered to 4 Somatic embryos are derived from callus (see Figure 2).

5) obtaining embryogenic callus from somatic embryos

When the somatic embryo obtained in 4) is cultured in the same medium as in 1), the embryogenic callus can be obtained again. You can repeat this method to get somatic embryos as you like.

6) Mass production of induced somatic embryos by seedlings in solid medium without growth regulator

Somatic embryos obtained in 4) and 5) do not germinate well in dormant state. Therefore, the somatic embryos are cold-treated at 0 ° C. for about 50 days, and then cultured in 1/3 murascige and squeegee medium without growth regulators to germinate as seedlings (see FIG. 5).

Example 1

1) inducing embryogenic callus from cultivated ginseng and wild cotyledon in medium without growth regulator

After aseptically treating mature ginseng seeds, the seeds are dissected to aseptically shed the zygote and then the cotyledons are cut. The cut cotyledons were adjusted to pH 5.8, the growth regulator was not added at all, and the concentration of Murashige and Squeegee medium was changed by 1 and 2 times and 3 times. Only one, two, three, four times different. All media are incubated in murascige and squeegee medium with 3% sugar and 0.8% agar. At this time growth chamber conditions are 24 ± 2 ℃, 16 hours and illuminated with 24μM m ^-2 s-1 white fluorescent lamp. In Squeegus medium supplemented with Murashige and Squeegee ammonium nitrate, the induction rate of embryoid callus was highest at the base of cotyledons after about 2 months (Table 1, Fig. 1). Callus was a mixture of fragile callus and hard callus, but uniform cell lines were obtained by subcultured 10 times at 5 week intervals in Squeeg medium containing twice the ammonium nitrate in the base of Murashige and Squeegee. Somatic embryos are also directly generated at the base of cotyledon in the basal medium of murascige and squeegee. If necessary, the somatic embryo can be propagated to obtain a seedling plant. In addition, the medium in which the concentration of ammonium nitrate increased by four times was very low in the induction rate of callus, and in the medium which increased by three times, the embryogenic callus was induced but browned after about two weeks. On the other hand, when ginseng cultivation is cultured in a medium having increased ammonium nitrate without cutting cotyledon in ginseng zygote, callus is formed on the cotyledon surface. If necessary, this callus can be used for the next step. In addition, germination of wild ginseng seeds induced the embryogenic callus by the same method, and the same result was obtained. Therefore, this method could be applied to both grown ginseng and natural wild ginseng.

2) culturing the induced embryogenic callus in a solid medium without the addition of a biomodulator

To investigate the concentration of murasigge and squag on the growth of embryogenic callus, the media concentration of murascige and squag is doubled, 1 times, 1/2 times, 1/3 times, 1/4 times, and ammonium nitrate 1 g of embryonic callus was subcultured to the solid medium and the liquid medium containing 10 mL of murascige and Squeeze medium without any growth regulator added. This cell line is browned and unable to grow due to the secretion of browning material from embryogenic callus from about 1 week of culture in the basal medium of murasigge and scog. However, the growth of the cell lines is maintained rapidly in the medium in which the Murashige and Squeege basal medium are lowered to 1/3 the concentration (see FIGS. 3 and 6). Repeated subculture to produce as many ginseng cell lines as desired. In addition, the concentration of sugar was changed to 1%, 3%, 6%, 6%, 9%, and 12% in 1/3 murashig Squeeg medium, and the concentration of sugar was 1% to 12%. As far as increases, the embryogenic cells are slightly white and present as spherical bumps. However, cell proliferation does not change significantly with sugar concentration.

3) suspending the induced embryogenic callus in a liquid medium not added with the life control agent

Into a 300 mL flask, add 100 mL of liquid medium containing no growth regulator with murascige and squeegee medium to 1/3 concentration, and reduce sugar concentration to 1%, and transfer 500 mg of embryogenic callus obtained in 2). A large amount of ginseng cell mass is produced by culturing at 100 revolutions per minute with a rotary shaker under about 500 lux or less. By repeatedly subcultured the obtained embryonic cell mass in the same manner, a large amount of the embryonic cell mass can be obtained as desired (see FIG. 4). In particular, the cell lines were grown in more than 20 liters of bioreactors, allowing for large tank cultures.

4) inducing somatic embryos from embryogenic callus induced in cotyledon

Embryonic callus obtained from the ginseng cotyledon was immediately transferred to a solid medium with pH lowered to 4 in normal myracige and squeeze medium containing 3% sugar and 0.8% agar, but without growth regulator. Somatic embryos are derived from callus (see FIG. 2).

5) obtaining embryogenic callus from somatic embryos

When the somatic embryo obtained in 4) is cultured in the same medium as in 1), the embryogenic callus can be obtained again. You can repeat this method to get somatic embryos as you like.

6) Mass production of induced somatic embryos by seedlings in solid medium without growth regulator

Somatic embryos obtained in 4) and 5) do not germinate well in dormant state. Therefore, the somatic embryos are cold-treated at 0 ° C. for about 50 days, and then cultured in 1/3 murascige and squeegee medium without growth regulators to germinate as seedlings (see FIG. 5).

According to the present invention, the ginseng cells and seedlings of cultivated ginseng and wild ginseng, which are very demanding in cultivation and growth conditions, can be aseptically produced in large quantities in a medium without growth regulators, and the development of processed foods or seedlings can be reproduced. Opened the way.

Claims (4)

Aseptic cutting of the cotyledons of seeds obtained by germinating ginseng seeds, and then doubled the concentration of ammonium nitrate and incubated in murascige and scoop solid medium without growth hormone at all to induce embryonic callus Ginseng cells comprising the steps of culturing the somatic cell embryo to obtain a uniform cell line, suspending the cell line in a solid medium and a liquid medium to obtain a large amount of ginseng cells, and producing germination and seedlings of the somatic cell embryo. And mass production of seedlings. The step of culturing the somatic embryo to obtain a uniform cell line according to claim 1, wherein the cultured cell line is passaged 10 times at intervals of 5 weeks in murasigge and squeegee medium without growth hormone at all to obtain a uniform cell line, and the cell line is ammonium nitrate. Method for producing a large amount of ginseng cells by repeating the subculture with a musiage and a squeegee medium without increasing the growth concentration and no growth hormone. The ginseng cell line obtained in claim 2 was reduced to 1% of the basic medium concentration of myracige and squeegee and reduced to 1% of sugar, suspended in solid medium and liquid medium without growth hormone at all, and repeated. Method for obtaining a large amount of ginseng cells The somatic embryo was subjected to low temperature treatment at 0 to 5 ° C. or to ₁ to 30 mg / L of GA ₃ to germinate in 1/3 murascige and squeegee medium without growth regulators to mass-produce seedlings. How to characterized.