KR100333558B1 - Mass propagation system of somatic embryo formation and their plant conversion by cell suspension culture in eleutherococcus senticosus maxim - Google Patents

Abstract

The present invention relates to a method for inducing cells aseptically from seeds of Eleutherococcus senticosus Maxim. And then producing a large amount of plants through somatic embryo development therefrom. Somatic embryos are derived from the cotyledons and embryogenic callus is derived from the somatic embryos, and then the callus is cultured in a solid or liquid medium to induce a large amount of somatic embryos and germinate to grow prickly tortilla plants.

Description

Somatic Embryogenesis and Plant Mass Production System by Cell Suspension Culture

The present invention relates to a method of inducing embryogenic cells capable of inducing a plant from prickly pears and culturing these cells to produce plants indefinitely in a culture vessel, in particular somatic cells directly from the surface of cotyledon of prickly pears. Embryonic callus is induced from these somatic embryos after induction of embryos. Thereafter, the callus is cultivated in a solid and liquid medium to produce a large amount of plants of prickly pears.

Eleutherococcus senticosus Maxim. Or Acanthopanax senticosus is a woody plant belonging to the Ogalpiaceae , which is used for medicinal root and bark for medicinal purposes (Brekhman, 1960, Izu Sibir Otdel. Akad. Nauk. USSR). In Russia, it is called Siberian ginseng (Lee, 1979, Korean Journal of Pharmocology) and has been shown to be effective in various fields such as anticancer, diabetes, arthritis and fatigue recovery (Lee, Sang-rae et al., 1989, Journal of Oriental Plant Botanicals 2: 1-). 214). Although it is designated as a legal information lake in the Soviet Union, China, Japan, and Korea, it is almost extinct due to the detriment of the deep mountains in Deogyusan, Jirisan, and Seorak. Seed germination requires ripening for more than three years in nature, and must be artificially stratified for more than 18 months (kuribayashi and Ohashi, 1971, Soyakugaku Zasshi 25: 95-101; Isoda and Shoji, 1994, Natural Medicine 48: 75-81) This treatment is difficult to germinate and difficult to breed. In the case of such a difficult breeding plant, the production of seedlings in large quantities using tissue culture techniques in a laboratory may be a significant breeding means.

The earliest report on the tissue culture of prickly pear is derived from the immature embryos and re-differentiated into plants (Gui et al., 1991, Plant cell Report 9: 514-516). Cattle (1993, Korean Journal of Plant Tissue Culture 20: 261-266) induced somatic embryos from zygotic embryos, but induces seedlings. In addition, Choi et al. (1997, Plant Cell Report 17: 84-88) reported the regeneration of a plant by somatic embryogenesis cell culture from island gallop, but island gallop is very well grown by seed breeding or cutting. Its use value is low because its effect is much lower than that of thorns.

The present invention induces somatic embryos directly from cultured tissues aseptically in a culture vessel, and then induces embryogenic callus from the somatic embryos to induce a large amount of plants by suspension culture or solid culture.

An object of the present invention is to provide a method for mass-producing plants by inducing embryonic cells from seed of T. aureus and culturing cells and callus in a culture vessel to mass produce and germinate somatic embryos.

The object of the present invention described above is to directly induce somatic embryos from seed-derived cotyledon of P. falciparum and induce embryogenic callus from the somatic embryos and to produce a large amount of somatic embryos through suspension culture or solid culture. This is achieved by the process of the invention for producing plants.

Figure 1 is a photograph of inducing somatic embryos from cotyledon seed-derived cotyledon.

Figure 2 is a photograph of inducing embryogenic callus from somatic embryos induced in cotyledons.

Figure 3 is a photograph of suspension culture of embryogenic cells

Figure 4 is a large amount of somatic embryos induced by cell suspension culture

5 is a germ cell germination

Fig. 6 is a photograph of plants Fig. 7 is a photo of prickly cultivated soil

The present invention is a method of inducing somatic embryos directly from cotyledon seed-derived cotyledon, inducing embryogenic callus from the somatic embryos, and inducing somatic embryos from the embryogenic callus, and then germinating them to mass produce prickly tortilla plants. It consists of.

The characteristics of the present invention are to induce somatic embryos directly from seed-derived cotyledons, to induce embryonic callus from induced somatic embryos to induce a large amount of somatic embryos, and to germinate these somatic embryos to mass produce plants.

In the method of the present invention, in order to produce a large number of prickly tortilla plants, somatic embryos are directly induced from cotyledon derived from prickly tortilla seeds, and the embryogenic callus is induced from the induced somatic embryos, and then these embryogenic callus are extracted from the liquid and Somatic embryos are obtained in solid medium in large quantities, and the somatic embryos obtained are germinated in a medium containing gibberellin, and the germinated seedlings can be transplanted into soil and produced as seedlings.

Therefore, the present invention is the primary somatic embryo induction step of directly inducing somatic embryos from cotyledon seed derived cotyledon, inducing embryogenic callus from induced somatic embryos, inducing somatic embryos from induced embryogenic callus, induction It also relates to a method of producing seedlings of pruning seedlings comprising the step of germinating germinated somatic embryos by gerberine treatment and transplanting the germinated plants into soil to produce seedlings.

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

1) Primary somatic embryo induction step of directly inducing somatic embryos from seed-derived cotyledons

When the seeds of P. falcipar were stratified at 10 ° C. for 18 months, the size of the embryo in the seed was about 7 mm. The seeds were sterilized and then aseptically removed from the zygote and germinated in 1/2 Murashige and Skoog`s, 1962, Physiologia Plantarum 15: 473-497 medium containing 0.7% agar. Induce. A week after germination, cotyledons of about 2 cm in length are cut and cultured in murascige and squeegee medium adjusted to pH 5.8 by adding 4.5 μM 2,4-D, 3% sugar and 0.7% agar. At this time, the culture room conditions are illuminated for 24 hours by 24 ± 2 ℃, 24μmol m ^-2 s ^-1 white fluorescent lamp. After about two months more than 30 somatic embryos are produced from one cotyledon (see Figure 1). However, the number of somatic embryos derived from the cotyledon of plants larger or smaller than 2 Cm in germination may be small (10 or less per cotyledon) or malformed embryos may be induced. If necessary, seedlings can be obtained by growing the somatic embryos described above.

2) inducing embryogenic callus from induced somatic embryos

Somatic embryos derived directly from cotyledon were cultured again with culture medium containing 4.5 μM 2,4-D such as the induction of primary somatic embryos and embryogenic callus was formed at the root of the embryo (Fig. 2). Reference).

3) inducing somatic embryos from induced embryogenic callus

The induced embryogenic callus is further subcultured at intervals of 5 weeks in the same medium containing 4.5 μM 2,4-D to maintain and propagate the embryogenic callus. The embryogenic callus was transferred to murascige and scoop solid medium containing 2,4-D and cultured to obtain more than 1600 somatic embryos per 500 mg of callus. In addition, the embryonic callus is subcultured with a liquid medium containing 4.5 μM 2,4-D for cell suspension culture and subcultured at intervals of 5 weeks to maintain and propagate the embryogenic callus (see FIG. 3). To generate somatic embryos, embryogenic callus can be passaged with murascige and squeegee liquid medium not containing 2,4-D to obtain more than 5,500 somatic embryos at 200 mg of callus (see FIG. 4). Therefore, by repeating this method using the induced somatic embryos, a large amount of somatic embryos can be obtained exponentially.

4) Germination of Induced Somatic Embryos and Production of Seedlings

Germ cell embryos obtained from embryogenic callus are not easy to germinate. Therefore, in the present invention, more than 95% of germination occurs when cultured in Murashige and Squeeged solid medium containing 3 μM GA ₃ , 0.7% agar and 3% sugar for induction of somatic embryos (see FIG. 5). Germinated seedlings are transferred to 1/3 murascige and squeegee solid medium containing 1% sugar and 0.7% agar to grow healthy plants (see FIG. 6). Plants can be purified from the soil and then produce young seedlings (see FIG. 7).

Example 1

Primary somatic embryo induction stage

When the seeds of prickly golia were stratified at 10 ° C. for 18 months, the size of the embryo in the seed was about 7 mm. The seeds were sterilized and then aseptically removed from the zygote to induce germination in 1/2 Murashige and Skoog's (Murashige and Skoog's, 1962, Physiologia Plantarum 15: 473-497) medium containing 0.7% agar. A week after germination, cotyledons were cut, and cultured in murascige and squeegee medium adjusted to pH 5.8 by adding 4.5 μM 2,4-D, 3% sugar and agar 0.7%. At this time, the culture room conditions were incubated with 24 ± 2 ° C., 24 μmol m ⁻² s ⁻¹ white fluorescent lamp for 16 hours, and somatic embryos were directly induced from cotyledons.

Embryonic callus induction stage

Somatic embryos directly derived from the cotyledons were cultured again under the same culture conditions and in the same medium as those inducing the primary somatic embryos to induce embryonic callus at the root of the embryo.

Inducing somatic embryos from embryogenic callus

The induced embryogenic callus was further subcultured at intervals of 5 weeks under the same culture conditions and the same medium as the embryogenic callus induction step to keep the embryogenic callus proliferated. These embryogenic callus were transferred to Murashige and Squeeged solid medium without 2,4-D to induce somatic embryos. In addition, the embryonic callus is subcultured with liquid medium containing 4.5 μM 2,4-D for cell suspension culture and passaged at intervals of 5 weeks to proliferate and maintain the embryogenic callus. Embryonic callus was passaged in Myracige and Squeeged liquid medium containing no 2,4-D to obtain somatic embryos.

Germination of Induced Somatic Embryos and Production of Seedlings

In order to germinate somatic embryos obtained from embryonic callus, more than 95% of germinated cells were cultured in Myracige and Scuba solid medium containing 3 μM gibberellin (GA ₃ ), 0.7% agar and 3% sugar. Germinated seedlings were transferred to 1/3 murascige and squeegee solid medium containing 1% sugar and 0.7% agar to grow into healthy seedlings.

According to the present invention, it is possible to produce a large number of saplings of prickly thorns, which are difficult to breed, and to supply the psylliums by seed production, thereby restoring the extinct organisms.

Claims (6)

The cotyledons of the seeds obtained by germinating seeds of prickly pears were cut and added with 4.5 μM 2,4-D, 3% sugar and 0.7% agar, and cultured in murascige and squeegee medium adjusted to pH 5.8 to directly somatic cells from cotyledons. Inducing embryos directly and culturing the somatic embryos and incubating again in a medium to which 4.5 μM 2,4-D is added, inducing embryogenic callus from somatic embryos, inducing somatic embryos from induced callus , A method of mass production of prickly pear plants, comprising the steps of producing a plant by germinating induced somatic embryos. Aseptic germination of cotyledons was added to 4.5 μM 2,4-D, 3% sugar, agar 0.7% and cultured in Murashige and Squeeged solid medium adjusted to pH 5.8 to obtain somatic embryos from the cotyledons surface. How to induce. The somatic embryo derived from claim 2 was added to 4.5 μM 2,4-D 3% sugar and 0.7% agar, and cultured in murasage and squeegee medium adjusted to pH 5.8 to culture embryonic callus from the root edge of the embryo. How to induce. A method for propagating and maintaining embryogenic callus by passage of embryogenic callus with murascige and squag solid and liquid medium added 4.5 μM 2,4-D, 3% sugar for cell suspension culture and adjusted to pH 5.8. The method of claim 1, wherein the embryogenic callus is derived from embryogenic callus by murascige and scoop solid medium or liquid medium containing 3% of sugar without 2,4-D. Method of obtaining somatic embryos with high frequency by culturing. The method of claim 1, wherein the induced somatic embryos are germinated, followed by producing the plants, wherein the somatic embryos are treated with 3 μM gibberellin (GA ₃ ) to germinate in murascige and squeegee solid medium to produce plants. Way.