KR19990025936A - Production Method of Ginseng Root Using Plant Tissue Culture - Google Patents

Production Method of Ginseng Root Using Plant Tissue Culture Download PDF

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KR19990025936A
KR19990025936A KR1019970047810A KR19970047810A KR19990025936A KR 19990025936 A KR19990025936 A KR 19990025936A KR 1019970047810 A KR1019970047810 A KR 1019970047810A KR 19970047810 A KR19970047810 A KR 19970047810A KR 19990025936 A KR19990025936 A KR 19990025936A
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ginseng
root
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ginseng root
saponin
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오훈일
이우진
김정혜
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0025—Culture media for plant cell or plant tissue culture
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones

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Abstract

본 발명은 식물조직배양방법에 의해 고려인삼의 root를 생산하여 인삼 Ext. 및 유효 약효성분인 사포닌을 생산하는 방법에 관한 것으로서, 수삼이나 고려인삼의 씨앗을 발아시켜 생육된 인삼 root로부터 사포닌 생성능력이 우수한 root를 유도하는 공정, 오옥신함량이 사이토키닌 함량에 비하여 상대적 과량인 식물성장호르몬 농도조성을 갖는 고려인삼 root배양용 고체배지를 사용하여 root를 유도하는 공정, 유도된 root를 오옥신함량이 사이토키닌 함량에 비하여 상대적 과량인 식물성장 호르몬 농도조성을 갖는 고려인삼 root배양용 액체배지에서 배양하여 root를 증식시키는 공정, 증식시켜 수확한 root를 오옥신함량이 상대적으로 많고 사이토키닌이 포함되어 있지 않으며, 인산염 농도가 감소하고 배양배지의 수소이온 농도가 증가한 액체배지에서 배양하여 사포닌 생성을 유도하는 공정을 포함하여 이루어지며 밭에서 재배한 인삼으로부터 인삼 Ext. 및 사포닌을 추출하는 종래의 기술이 가지고 있던 문제점들을 해결하고, 다량의 인삼 Ext. 및 사포닌을 단기간에 얻을 수 있는 식물조직배양 방법에 의한 고려인삼 root의 생산방법에 관한 것이다.The present invention is to produce the root of Korean ginseng by plant tissue culture method Ginseng Ext. And a method for producing saponin, an active drug, which is derived from the ginseng root of ginseng or Korean ginseng, which has excellent saponin-producing ability, and has a relative excess of oxine content compared to cytokinin content. A process for inducing roots using a solid medium for Korean ginseng root culture having a composition of phosphorus plant growth hormone concentration, and for the culture of Korean ginseng roots having a plant growth hormone concentration composition in which the induced root has an excessive amount of oxine compared to the cytokinin content The process of propagating root by culturing in liquid medium, and the root harvested by multiplying is grown in a liquid medium with a relatively high oxine content and no cytokine, phosphate concentration is decreased, and hydrogen ion concentration of culture medium is increased. Phosphorus grown in the field, including processes that induce saponin production From Ginseng Ext. And solve the problems with the prior art of extracting saponin, and a large amount of Ginseng Ext. And it relates to a production method of Korean ginseng root by the method of plant tissue culture that can obtain saponin in a short time.

Description

식물조직배양을 이용한 인삼근의 생산방법Production Method of Ginseng Root Using Plant Tissue Culture

본 발명은 식물조직배양방법에 의한 고려인삼(Panax ginseng C.A. Meyer) 근(root)의 생산방법, 보다 자세하게는 고려인삼에 들어있는 유효 약효성분인 사포닌 및 기타 유효성분을 식물근 배양법에 의해 생산하는 방법에 관한 것이다.The present invention is a method of producing root ginseng (Panax ginseng CA Meyer) root by plant tissue culture method, more specifically, saponin and other active ingredients contained in Korean ginseng by plant root culture method It is about a method.

고려인삼(Panax ginseng C.A. Meyer)은 반음지성 숙근초로서 오가피과(Araliaceae) 인삼속에 속하는 다년생 식물로 인삼의 약리적 효능은 건강한 사람에게는 항상성을 유지해 주며 병약자에게는 항상성을 정상화시켜 준다. 이에 대해 과학적으로 입증되고 있는 약리효과를 보면 간기능 보호작용, 항당뇨기능, 심장강화 및 혈압조절, 항암효과, 뇌기능 강화, 면역기능 강화 등이 있다. 인삼 사포닌은 인삼속 식물에만 함유된 특이한 모형의 담마레인(dammarane)계 트리터펜(triterpene) 배당체로 ginsenoside라고 한다. 인삼 사포닌은 사포제닌(sapogenin)인 프로토파낙사디올(protopanaxadiol)과 프로토파낙사트리올(protopapanaxatriol)의 알콜성 -OH기에 글루코스(glucose), 람노스(rhamnose), 자일로스(xylose), 아라비노스(arabinose) 등과 같은 당류가 에테르 결합된 것으로 고려인삼에서는 총 26종의 구조가 밝혀졌다. 사포닌은 인삼 뿐만 아니라, 다른 여러 식물에도 함유되어 있는데, 인삼의 경우 주요 약효성분으로 작용을 하지만 식물에 따라 유해성분으로 작용하는 것도 있다. 인삼 엑기스(extract) 및 사포닌은 다년생 식물인 인삼을 재배하여 수확, 추출하여 제품화하고 있는데 기존의 밭에서 재배한 인삼으로부터 엑기스 및 사포닌을 추출하는 방법은 다음과 같은 단점이 있다.Korean ginseng (Panax ginseng C.A. Meyer) is a perennial plant belonging to the genus Araliaceae of ginseng, and its pharmacological effects maintain homeostasis in healthy people and normalize homeostasis in sick people. Scientifically proven pharmacological effects include liver function protection, antidiabetic function, heart strengthening and blood pressure control, anticancer effect, brain function enhancement, and immune function enhancement. Ginseng saponin is a unique type of dammarane triterpene glycoside that is contained only in the genus Ginseng. It is called ginsenoside. Ginseng saponins are alcoholic -OH groups of saponogenin (protopanaxadiol) and protopapanaxatriol (glucose, rhamnose, xylose, arabinose). Sugars such as arabinose and the like were ether-linked, and a total of 26 structures were identified in Korean ginseng. Saponin is contained not only in ginseng, but also in many other plants. Ginseng acts as a major medicinal ingredient, but some plants act as harmful components. Ginseng extract (extract) and saponins are grown, harvested, extracted and commercialized perennial plant ginseng, the method of extracting the extract and saponins from the ginseng grown in the existing field has the following disadvantages.

첫째. 인삼의 생육기간이 4-6년으로 길며 6년간 재배해야 100-150g(fresh wt)의 수삼을 수확할 수 있다.first. The growth period of ginseng is 4-6 years long and it needs to be grown for 6 years so that 100-150g (fresh wt) of ginseng can be harvested.

둘째. 연작이 불가능하여 재배면적이 줄어들고 있으며 해가림의 특수한 시설조건 하에서만 재배가 가능하다.second. It is not possible to grow, so the area of cultivation is decreasing, and it is possible to grow only under special facility conditions of sunburn.

셋째. 재배조건이 까다롭고 기후, 토양, 병충해 등 환경의 지배를 많이 받아 공급이 안정되지 못하다.third. The growing conditions are difficult and the supply is not stable due to the environment, climate, soil and pests.

넷째. 인삼은 이식법을 이용하여 다년간 재배해야 하므로 병충해 예방을 위하여 농약을 사용하기 때문에 농약잔류의 가능성이 높다.fourth. Since ginseng must be grown for many years using transplantation methods, pesticide residues are high because pesticides are used to prevent pests.

위와 같이 밭에서 재배한 인삼에서 엑기스 및 사포닌을 생산하는데 발생하는 문제를 해결하기 위하여 최근 세포배양에 의해 생산된 인삼캘러스를 재배인삼 대용으로 사용하기 위한 연구가 꾸준히 진행되어 왔다.In order to solve the problems caused by the production of extract and saponin from the ginseng cultivated in the field as described above, the research to use the ginseng callus produced by cell culture as a substitute for cultivated ginseng has been steadily progressed.

그러나, 식물세포배양은 종종 필요성분의 함량이 낮고 생산성의 불안정성 등 몇가지 문제들에 접하게 되는데, 이러한 문제점들을 극복하기 위하여 최근에는Agrobacterium rhizogenes감염에 의한 모상근(hairy root) 배양에 대해 관심을 기울이고 있다. 모상근을 이용한 현재까지의 연구에서는 모상근 유도시 토양세균인Agrobacterium rhizogenes를 감염시켜 형질전환을 일으키도록 하여 모상근을 유도하였는데(일본특허공보 제 소63-254982, 일본특허공보 제 평3-285,690호, 일본특허공보 제 평4-341194호), 형질전환에 의해 모상근을 유도시킬 경우 형질전환을 위해 감염시킨 균을 제거하기 위해서 반코마이신(vancomycin), 카베니실린(carbenicillin), 테트라싸이클린(tetracycline) 등과 같은 항생제를 사용해야 하며, 균을 제거하는데 많은 시간이 필요하고, 균을 죽인 뒤에 다시 배지를 바꾸어주어야 하는 등의 번거로운 과정을 거쳐야 하는 단점이 있다. 또한 외래 유전자인 Ri 플라스미드(Ri plasmid)가 인삼염색체에 무작위로 혼입되므로 이로 인한 인삼유전자의 발현(gene expression)이 달라져 인삼엑기스의 경우 안전성에 대한 검토가 있어야 할 것으로 사료된다.However, plant cell culture often encounters several problems such as low content of necessary ingredients and instability of productivity. Recently, attention has been paid to hairy root culture by Agrobacterium rhizogenes infection to overcome these problems. To date, studies using hairy roots have induced hairy roots by inducing transformation by infecting soil bacteria Agrobacterium rhizogenes during hairy root induction. Patent Publication No. Hei 4-341194), in the case of inducing hairy roots by transformation, antibiotics such as vancomycin, carbenicillin, tetracycline, etc. It has to be used, it takes a lot of time to remove the bacteria, and has to go through a cumbersome process, such as changing the medium after killing the bacteria. In addition, since the foreign gene Ri plasmid is randomly mixed into the ginseng chromosome, the gene expression of the ginseng gene is changed. Therefore, the safety of the ginseng extract should be considered.

본 발명자들은 여러가지 방법으로 연구를 거듭한 결과, 지금까지 국내외 학계에서 성공사례가 없었던 균의 감염없이 유도된 인삼근t 배양에 의해서 인삼 엑기스 및 사포닌을 생산하는 방법을 개발하여 본 발명을 완성하였다.As a result of repeated studies in various methods, the present inventors have developed a method for producing ginseng extract and saponin by ginseng root culture, which is induced without infection of bacteria that have not been successful in domestic and foreign academia.

따라서, 본 발명의 목적은 밭에서 재배한 인삼으로부터 인삼 엑기스 및 사포닌을 추출하는 종래의 기술이 가지고 있던 문제점들을 해결하고, 다량의 인삼 엑기스 및 사포닌을 단기간에 얻을 수 있는 식물조직배양 방법에 의한 고려인삼근의 생산방법을 제공함에 있다.Accordingly, an object of the present invention is to solve the problems of the conventional techniques of extracting ginseng extract and saponin from ginseng grown in the field, and to consider the ginseng by plant tissue culture method that can obtain a large amount of ginseng extract and saponin in a short period of time. To provide a production method of triceps.

즉, 본 발명은 ⅰ) 인삼의 씨앗을 발아시키거나 수삼의 절편으로부터 유도된 캘러스로부터 근형성이 우수한 세포를 유도하는 살균 및 캘러스 유도공정 ; ⅱ) 오옥신의 함량이 사이토키닌의 함량에 비하여 상대적 과량인 식물성장 호르몬을 갖는 인삼근 유도용 고체배지를 사용하여 근형성 능력이 우수한 세포를 유도하는 인삼근의 유도공정 ;That is, the present invention is a bactericidal and callus induction process for germinating seeds of ginseng or inducing cells excellent in muscle formation from callus derived from slices of ginseng; Ii) the process of inducing ginseng root which induces cells having excellent muscle formation ability by using a ginseng root-derived solid medium having a plant growth hormone with an excessive amount of oxine relative to the content of cytokinin;

ⅲ) 유도된 근을 오옥신의 함량이 사이토키닌의 함량에 비해 상대적 과량인 식물성장 호르몬 농도를 갖는 인삼근 배양용 액체배지에서 인삼근을 유도시키는 인삼근의 증식배양 공정 ; 및Iii) Proliferation and cultivation process of ginseng root to induce ginseng root in liquid culture medium for ginseng root cultivation, which has a concentration of plant growth hormone which is relatively higher than that of cytokinin; And

ⅳ) 증식시켜 수확한 인삼근을 오옥신함량이 높고 사이토키닌을 포함하지 않은 액체배지에서 배양하여 사포닌(saponin) 및 기타 인삼성분 생성을 유도하는 사포닌 유도공정을 포함하는 식물조직배양을 이용한 인삼근의 생산방법 임을 특징으로 한다.인) Ginseng root, which is grown by incubating saponin and other ginseng components by cultivating the ginseng roots grown by multiplying in a liquid medium containing high oxine and not containing cytokinin Characterized in that the production method.

본 발명에서는 먼저 고려인삼의 씨앗이나 수삼을 살균하고 멸균증류수로 헹군 뒤 식물성장호르몬이 첨가된 발아용 한천 평판배지에 심어서 암조건으로 3개월간 배양하여 발아시키거나 근을 유도한다. 발아 후, 근생장이 활발한 세포주를 선발하여 오옥신 함량이 사이토키닌 함량에 비해 상대적으로 과량인 식물성장호르몬 농도조성을 갖는 배지에 옮겨 계대배양하면 근생장율이 높은 세포주가 유도된다.In the present invention, first, sterilize the seeds or ginseng of Korean ginseng, rinse with sterile distilled water, and then plant in agar plate medium for germination with plant growth hormone added to incubate for 3 months under dark conditions to induce germination or muscle. After germination, cell lines with active myocyte growth are selected and transferred to a medium having a plant growth hormone concentration composition with a large amount of oxine compared to the cytokinin content, and subcultured to induce a high cell growth rate.

위와 같이하여 유도된 근을 오옥신 함량이 사이토키닌 함량에 비해 상대적으로 과량인 식물성장호르몬 농도조성을 갖고, 자당, 질소원, 인산염이 첨가된 근배양용 액체배지에 접종하여 25℃ 배양기에서 암조건으로 근을 증식시켜 이들 근을 수확한다.The roots derived as above were inoculated in a liquid growth medium containing a large amount of plant growth hormone with a relatively high concentration of oxine compared to the cytokinin content, and inoculated in a liquid culture medium containing sucrose, a nitrogen source and a phosphate, and the roots were treated under dark conditions in a 25 ° C. incubator. Multiply to harvest these roots.

위와같이 증식된 근으로부터 사포닌 형성을 유도하기 위하여 인삼근 배양용 배지조성중 인산칼륨(potassium phosphate)와 황산암모늄(ammonium sulfate)를 사용하여 질소원의 농도를 조정하고, 칼륨염을 사용하여 인산염의 농도를 조정, 에너지원으로 사용되는 자당(sucrose)의 농도를 조정하며, 식물성장 호르몬인 오옥신을 첨가, 배지내 수소이온 농도를 조정하여 제조된 배양배지에서 증식되어 수확된 근을 25℃ 배양기에서 암조건으로 4주간 배양하여 사포닌을 유도하여 인삼 근으로 부터 사포닌을 얻는다.Potassium phosphate and ammonium sulfate were used to adjust the concentration of nitrogen sources in the culture of ginseng root culture to induce saponin formation from the proliferated muscles as described above. Adjust the concentration of sucrose used as energy source, adjust the concentration of hydrogen ions in the medium by adding oxine, a plant growth hormone, and adjust the concentration of hydrogen ions in the medium. Incubate for 4 weeks to induce saponins to obtain saponins from the ginseng root.

이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다. 그러나, 본 발명이 이들 실시예에 국한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

(실시예 1) 살균 및 캘러스 유도 공정Example 1 Sterilization and Callus Induction Process

고려인삼의 씨앗을 개갑한 것이나, 수삼의 절편을 70% 에탄올 용액과 4% NaOCl 용액으로 살균한 뒤 멸균 증류수로 세척한 다음 하기의 표 1과 같은 조성의 평판배지에 식물성장호르몬으로서 키네틴 1-(Kinetin)과 1-나프탈렌아세틱에시드(NAA), 6-벤질아미노퓨린(BAP)과 인돌아세틱에시드(IAA), 그리고 6-벤질아미노퓨린과 1-나프탈렌아세틱에시드 조합으로 첨가된 캘러스 유도용 한천배지에 심어 25℃의 배양기에서 암조건으로 3개월간 배양시켜 캘러스를 유도시켰다.The seeds of Korean ginseng were re-opened, and sections of fresh ginseng were sterilized with 70% ethanol solution and 4% NaOCl solution, washed with sterile distilled water, and then kinetin 1- as plant growth hormone on a plate medium of the composition shown in Table 1 below. (Kinetin) and 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP) and indoleacetic acid (IAA), and callus induction added with 6-benzylaminopurine and 1-naphthaleneacetic acid combination Planted in agar agar medium and incubated for 3 months in a dark condition in a 25 ℃ incubator to induce callus.

(실시예 2) 인삼근 유도 공정Example 2 Ginseng Muscle Induction Process

실시예 1에서 유도된 캘러스를 하기의 표 2와 같은 조성의 배지에 식물성장 호르몬으로서 키네틴(Kinetin)과 1-나프탈렌아세틱에시드, 6-벤질아미노퓨린과 인돌아세틱에시드, 그리고 6-벤질아미노퓨린과 1-나프탈렌아세틱에시드 조합으로 첨가된 한천배지에 심어 25℃의 배양기에서 암조건으로 3개월간 배야시켜 근을 유도시켰다.Callus derived from Example 1 was kinetin and 1-naphthaleneacetic acid, 6-benzylaminopurine and indoleacetic acid, and 6-benzylamino as plant growth hormones in a medium having a composition as shown in Table 2 below. Muscle was induced by incubating for 3 months under dark conditions in an incubator at 25 ° C. in agar medium added with a combination of purine and 1-naphthaleneacetic acid.

(실시예 3) 인삼근의 증식배양 공정Example 3 Growth Process of Ginseng Root

실시예 2에서 유도된 생장이 활발한 근을 표 2의 배지에 식물성장 호르몬으로서 6-벤질아미노퓨린과 1-나프탈렌아세틱에시드가 첨가된 액체배지에 옮겨 25℃ 원심회전 진탕배양기에서 암조건, 60 rpm 조건으로 배양하여 4주간격으로 계대배양 하였는데, 선택된 세포주의 배증시간(doubling time)은 약 10일이었다.The active growth induced in Example 2 was transferred to a liquid medium to which 6-benzylaminopurine and 1-naphthaleneacetic acid were added as plant growth hormones to the medium of Table 2, and the dark conditions in a 25 ° C centrifugal shaking incubator were 60. Cultured at rpm and subcultured at 4 week intervals, the doubling time of the selected cell lines was about 10 days.

[표 1] 배지의 조성TABLE 1 Composition of medium

[표 2] 배지의 조성TABLE 2 Composition of medium

(실시예 4) 인삼 사포닌 유도배양 공정(2차 대사산물의 유도 공정)Example 4 Ginseng saponin induction culture process (secondary metabolite induction process)

실시예 3에서 증식하여 수확한 인삼 근으로부터 사포닌을 유도하기 위하여, 실시예 2에서의 표 2와 같은 조성의 배지 1ℓ에 자당(sucrose), NH4H2PO4, 질소원, 오옥신을 첨가하고, 배지의 수소이온농도를 조절하여 제조한 인삼 사포닌 유도용 액체배지에 실시예 3에서 증식시켜 수확한 근을 1.5%씩 접종하여 25℃ 원심회전 진탕배양기에서 암조건, 60 rpm으로 4주ㅈ간 배양한 결과, 생체중량(fresh weight) 2.69g 을 얻어 배양초기보다 약 4.5배 생장하였고, 사포닌 함량은 0.27%로 총사포닌 함량은 7.26mg 이었다.In order to induce saponins from the ginseng roots grown and harvested in Example 3, sucrose, NH 4 H 2 PO 4 , a nitrogen source, and oxine are added to 1 L of a medium having the composition shown in Table 2 in Example 2, Ginseng saponin-derived liquid medium prepared by adjusting the hydrogen ion concentration of the medium was inoculated in 1.5% each of the roots harvested by growing in Example 3, and cultured for 4 weeks at 60 rpm in a dark condition at 25 ° C centrifugal shaking incubator. As a result, a fresh weight of 2.69 g was obtained, which was about 4.5 times larger than the initial culture, and the saponin content was 0.27% and the total saponin content was 7.26 mg.

위의 실시예와 같은 방법으로 인삼의 근을 배양하여 얻은 사포닌을 정량분석한 결과 도 1과 같은 결과를 얻었으며, 본 발명에 의한 사포닌 성분은 5년근 재배인삼의 사포닌 성분과 일치하는 인삼 사포닌 임을 확인하였다.As a result of quantitative analysis of saponins obtained by culturing ginseng roots in the same manner as in the above example, the results were obtained as shown in FIG. Confirmed.

본 발명의 생산방법에 의하면 균의 감염없이 유도된 인삼근 배양에 의해서 단기간에 기후, 토양, 병충해 등 자연환경의 지배를 받지 않고 고함량의 사포닌 및 인삼엑기스를 안정적으로 생산할 수 있다.According to the production method of the present invention it is possible to stably produce a high content of saponin and ginseng extracts without being controlled by the natural environment such as climate, soil, pests by the ginseng root culture induced without bacteria infection.

Claims (5)

ⅰ) 인삼의 씨앗을 발아시키거나 수삼의 절편으로부터 유도된 캘러스로부터 근형성이 우수한 세포를 유도하는 살균 및 캘러스 유도공정 ; ⅱ) 오옥신의 함량이 사이토키닌의 함량에 비하여 상대적 과량인 식물성장 호르몬을 갖는 인삼근 유도용 고체배지를 사용하여 근형성 능력이 우수한 세포를 유도하는 인삼근의 유도공정 ;Iii) sterilization and callus induction process of germinating seeds of ginseng or inducing cells having good muscle formation from callus derived from slices of ginseng; Ii) the process of inducing ginseng root which induces cells having excellent muscle formation ability by using a ginseng root-derived solid medium having a plant growth hormone with an excessive amount of oxine relative to the content of cytokinin; ⅲ) 유도된 근을 오옥신의 함량이 사이토키닌의 함량에 비해 상대적 과량인 식물성장 호르몬 농도를 갖는 인삼근 배양용 액체배지에서 인삼근을 유도시키는 인삼근의 증식배양 공정 ; 및Iii) Proliferation and cultivation process of ginseng root to induce ginseng root in liquid culture medium for ginseng root cultivation, which has a concentration of plant growth hormone which is relatively higher than that of cytokinin; And ⅳ) 증식시켜 수확한 인삼근을 오옥신함량이 높고 사이토키닌을 포함하지 않은 액체배지에서 배양하여 사포닌(saponin) 및 기타 인삼성분 생성을 유도하는 사포닌 유도공정을 포함하는 식물조직배양을 이용한 인삼근의 생산방법인) Ginseng root, which is grown by incubating saponin and other ginseng components by cultivating the ginseng roots grown by multiplying in a liquid medium containing high oxine and not containing cytokinin Production method 제 1항에 있어서, 캘러스 유도 공정은 식물성장호르몬조합을 갖는 인삼근 배양용 한천 평판배지에 유도된 근형성능이 우수한 캘러스를 접종하는 단계, 암조건에서25℃, 4주간 배양하는 연속적인 단계를 포함함을 특징으로 하는 식물조직배양법을 이용한 인삼근의 생산방법According to claim 1, Callus induction process is a step of inoculating the callus having excellent muscle formation ability induced in agar plate medium for cultivating ginseng root having a plant growth hormone combination, the continuous step of incubating for 4 weeks at 25 ℃ in dark conditions Production method of ginseng root using plant tissue culture method characterized by including 제 1항에 있어서, 인삼근의 증식배양 공정은 근유도능이 활발하여 선발된 세포주를 오옥신함량이 사이토키닌 함량에 비해 상대적 과량인 식물성장 호르몬을 갖는 근배양용 액체배지에 접종하는 단계, 25℃, 암배양의 조건으로 원심회전 배양에서 60 rpm, 4주간 배양시키는 연속단계를 포함함을 특징으로 하는 식물조직배양법을 이용한 인삼근의 생산방법According to claim 1, The growth process of ginseng root is inoculated into the liquid culture medium for muscle culture with plant growth hormone that the oxin content relative to the cytokinin content of oxine content selected by active muscle induction activity, 25 ℃ , Ginseng root production method using a plant tissue culture method characterized in that it comprises a continuous step of incubating for 4 weeks at 60 rpm, centrifugal rotation culture under the conditions of cancer culture 제 1항에 있어서, 사포닌 유도공정은 인삼근 배양용 액체배지에 증식되어 수확된 근세포를 오옥신 함량이 상대적으로 많고 인산염 농도가 감소되고, 수소이온농도가 증가된 새로운 배지로 바궈주는 단계, 25℃, 암배양의 조건으로 원심회전 배양에서 60 rpm, 4주간 배양시키는 연속단계를 포함함을 특징으로 하는 식물조직배양법을 이용한 인삼근의 생산방법The method according to claim 1, wherein the saponin induction process is performed by propagating the harvested myocytes into a liquid medium for ginseng root culture to a new medium having a relatively high oxine content, a reduced phosphate concentration, and an increased hydrogen ion concentration, at 25 ° C. , Ginseng root production method using a plant tissue culture method characterized in that it comprises a continuous step of incubating for 4 weeks at 60 rpm, centrifugal rotation culture under the conditions of cancer culture 제 4항에 있어서, 증식된 근으로 부터 사포닌 형성을 유도하기 위하여 인삼근 배양용 배지조성중 인산칼륨(potassium phosphate)와 황산암모늄(ammonium sulfate)를 사용하여 질소원의 농도를 조정하고, 칼륨염(potassium salt)을 사용하여 인산염의 농도를 조정하며 에너지원으로 사용되는 자당(sucrose)의 농도를 조정함을 특징으로 하는 식물조직배양법을 이용한 인삼근의 생산방법The method according to claim 4, wherein in order to induce saponin formation from the proliferated muscle, the concentration of the nitrogen source is adjusted using potassium phosphate and ammonium sulfate in the composition of the culture medium for the ginseng root, and the potassium salt (potassium) Method of producing ginseng root using plant tissue culture method characterized by adjusting the concentration of phosphate using salt) and adjusting the concentration of sucrose used as energy source
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