KR20030063724A - Production method of Callus and Adventitious root from the leaves and stems of ginseng - Google Patents

Production method of Callus and Adventitious root from the leaves and stems of ginseng Download PDF

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KR20030063724A
KR20030063724A KR1020020003973A KR20020003973A KR20030063724A KR 20030063724 A KR20030063724 A KR 20030063724A KR 1020020003973 A KR1020020003973 A KR 1020020003973A KR 20020003973 A KR20020003973 A KR 20020003973A KR 20030063724 A KR20030063724 A KR 20030063724A
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callus
ginseng
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root
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손성호
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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
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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
    • A01H4/002—Culture media for tissue culture
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/06—Roots
    • 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
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    • C12N2500/30—Organic components
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    • 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

PURPOSE: A method for production of the callus and adventitious root from the leaves and stems of ginseng is provided, thereby mass-producing the callus and adventitious root from the leaves and stems of ginseng. CONSTITUTION: A method for production of the callus and adventitious root from the leaves and stems of ginseng comprises the steps of: sterilizing the surface of leaves and stems of ginseng; penetrating the plant growth regulating material dissolved in medium into the surface sterilized leaf and stem tissue of ginseng; inducing callus and adventitious root from the leaves and stems of ginseng on the solid medium; and culturing the induced callus and adventitious root of ginseng in the liquid medium, wherein the sterilization is carried out by treating the surface of leaves and stems of ginseng with both 1 to 4% hypochlorous acid solution and 1 to 4% hydrogen peroxide.

Description

인삼류의 잎과 줄기를 이용한 캘러스 및 부정근 유도와 그 증식방법 {Production method of Callus and Adventitious root from the leaves and stems of ginseng}Callus and Adventitious Root Using the Leaves and Stems of Ginseng Roots and Production Methods {Production method of Callus and Adventitious root from the leaves and stems of ginseng}

본 발명은 인삼류(인삼, 장뇌삼 및 산삼 등)의 잎과 줄기에 다량으로 존재하는 트리올(triol) 계통의 인삼 사포닌 등을 배양기 내에서 대량으로 생산하기 위하여, 잎과 줄기 조직으로부터 캘러스와 부정근을 유도한 후 이를 대량 복제하는 것에 관한 것이다. 특히 대량복제의 경우 액체의 영양액을 사용하여 무균적으로 배양함으로써 포지에서 재배되는 인삼류에 비해 농약성분이 전혀 없으며, 환경오염의 영향을 받지 않음으로 청정한 식품으로 사용할 수 있는 장점을 지니고 있다. 특히 배양기 내에서 식물의 세포 및 조직을 복제하는데 주로 사용되는 2,4-D라는 인체에 유해한 인공 식물호르몬을 사용하지 않음으로써 기존의 조직배양에 의한 인삼류 생산기술에 비해 진일보된 것으로 생각된다.The present invention, in order to produce a large amount of triol ginseng saponins, such as triols present in the leaves and stems of ginseng (Ginseng, camphor and wild ginseng) in a large amount in the incubator, callus and intestinal roots from leaf and stem tissue It's about inducing and then mass cloning. Particularly, in the case of mass replication, aseptic culture using liquid nutrient solution, there is no pesticide component compared to ginseng grown in Forge, and it has the advantage of being used as a clean food because it is not affected by environmental pollution. In particular, 2,4-D, which is mainly used to replicate the cells and tissues of plants in the incubator, does not use artificial plant hormones that are harmful to the human body.

본 발명은 야외에서 자란 인삼류(인삼, 장뇌삼 및 산삼 등)의 잎과 줄기를 재료로 하여 조직배양에 의해 캘러스와 부정근을 대량으로 생산하는 방법에 관한 것이다.The present invention relates to a method for producing a large amount of callus and intestinal root by tissue culture using leaves and stems of ginseng (ginseng, camphor and wild ginseng) grown outdoors.

지금까지 인삼류는 뿌리조직을 이용하여 캘러스나 부정근을 증식하여 식품에 사용하려는 시도가 있었으며, 일본 일동정공의 경우 인삼의 뿌리조직 세포를 증식하여 최근에 바이오 식품을 일본시장에 출시한 바가 있으며, 우리나라에서도 인삼류의 뿌리 조직을 이용하여 캘러스나 부정근을 유도한 다음, 이를 증식하는 연구결과가 각종 논문이나 특허로 출원된 바 있다.Until now, ginseng has tried to use it in food by using the root tissues to grow callus or intestinal roots.In the case of Japan Ildong Precision, it has recently introduced bio foods to the Japanese market by proliferating the root tissue cells of ginseng. In the case of using the root tissue of ginseng to induce callus or inferior root, and then proliferate it, the research results have been applied to various papers or patents.

그러나 복제된 세포나 조직에서 생산되는 물질의 종류나 양은 원래 재료로 사용한 조직의 영향을 많이 받는 것으로 알려져 있음에도 불구하고, 뿌리에 비해 특히 트리올(triol) 계통의 함량이 높은 잎과 줄기 등을 이용하여 이로부터 세포나 조직을 유도한 후, 증식하여 사용하려는 시도는 전세계적으로도 거의 연구되지 못하고 있는 실정이다. 그 원인으로는 자연상태에서 자란 잎과 줄기 조직은 조직배양을 하기 위해 잎 표면이나 조직내부의 감염되어 있는 미생물을 완전히 제거하기가 어렵다는 점을 들 수 있다.However, despite the fact that the type and amount of substances produced from cloned cells or tissues are influenced by the tissues used as the original materials, the leaves and stems, which have a higher content of the triol strain than the roots, are used. Therefore, attempts to proliferate and use cells after inducing cells or tissues have been rarely studied in the world. The reason is that it is difficult for the leaves and stem tissues grown in nature to completely remove the infected microorganisms in the leaf surface or inside the tissue for culture.

본 발명의 목적은 인삼류의 잎과 줄기부분에 함유되어있는 특정성분을 대량으로 얻기 위하여 인삼류의 잎과 줄기 조직을 재료로 하여, 이로부터 캘러스나 부정근을 대량생산하여 궁극적으로는 그 배양산물을 식품이나 의약품 원료로 사용할 수 있는 최적의 방법을 개발하는 데 있다.An object of the present invention is to make a large amount of specific components contained in the leaves and stems of ginseng in order to produce a large amount of callus or root muscle from the leaves and stem tissue of the ginseng, and ultimately the culture product However, it is to develop an optimal method that can be used as a raw material for pharmaceuticals.

특히 본 발명의 또 다른 목적은, 인삼류의 연약한 잎조직과 줄기의 형성층 조직의 표면 소독을 위해 소독제의 선택과 효과적이고도 재현성이 있는 제균 방법의 개발 및 조직내부로 식물생장조절물질을 직접 흡수시켜 줌으로써 캘러스나 부정근을 단시일 내에 효과적으로 유도시킬 수 있는 방법을 제공하는 데 있다.In particular, another object of the present invention is to select a disinfectant and develop an effective and reproducible bactericidal method for the surface disinfection of soft leaf tissues and stem-forming tissues of ginseng and by directly absorbing plant growth regulators into the tissue. The present invention provides a method for effectively inducing callus or abscess in a short time.

제1도는 인삼류 잎조직으로부터 부정근이 유도되는 모습으로, 도 1a는 인삼,1 is a state in which the root of the ginseng is derived from the ginseng leaf tissue, Figure 1a ginseng,

도 1b는 장뇌삼, 도 1c는 산삼 시료의 모습을 찍은 사진이다.Figure 1b is a camphor ginseng, Figure 1c is a photograph taken the state of wild ginseng samples.

제2도는 인삼류의 줄기조직을 약 6주간 배양하였을 때 캘러스와 부정근이 동시에 유도되는 모습으로, 도 2a는 인삼, 도 2b는 장뇌삼, 도 2c는 산삼 시료의 모습을 찍은 사진이다.Figure 2 is a state in which the callus and the root of the ginseng are induced at the same time when the stem tissue of the ginseng stem cultured for about 6 weeks, Figure 2a is a ginseng, Figure 2b is a camphor ginseng, Figure 2c is a picture of a wild ginseng sample.

제3도는 급속생장을 하는 인삼류의 캘러스와 부정근의 사진으로, 도 3a는 캘러스를, 도 3b는 부정근의 모습을 찍은 사진이다.FIG. 3 is a photograph of callus and labia root of ginseng growing rapidly. FIG. 3A is a callus and FIG.

본 발명은 인삼류의 잎과 줄기의 형성층을 이용하여 캘러스와 부정근을 유도하는 것을 특징으로 하는 캘러스 및 부정근 증식방법에 관한 것이다.The present invention relates to a method of growing callus and root muscles, which induces callus and root muscles by using the cambium's leaf and stem forming layers.

구체적으로는, 본 발명은 야외에서 자란 인삼류(인삼, 장뇌삼 및 산삼 등)를 수집한 다음 잎과 줄기 조직을 분리하여 표면소독을 하는 단계, 배양액 속에 녹아있는 식물생장조절물질을 표면소독된 잎과 줄기 조직으로 침투시키는 단계, 고체배지에서 캘러스와 부정근을 유도시키는 단계 및 유도된 캘러스와 부정근을 대량증식하는 액체현탁배양 단계를 포함하는 것을 특징으로 한다.Specifically, the present invention collects ginseng (ginseng, camphor ginseng and wild ginseng, etc.) grown outdoors, and then disinfecting the leaves and stem tissue for surface disinfection, the plant growth regulator dissolved in the culture medium and the surface disinfected leaves and Penetrating into the stem tissue, inducing callus and involuntary muscle in a solid medium, and liquid suspension culture step of mass growth of the induced callus and inferior muscle.

이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.

배양하고자 하는 인삼류는 영주농업기술센타, 심마니협회 및 한국산삼협회로부터 인증 받은 인삼, 장뇌삼 및 산삼을 이용하였으며, 배양하고자 하는 조직의 표면소독 시, 소독 액의 농도가 높을 경우 조직이 파괴되고, 소독액의 농도가 낮을 경우 각종 곰팡이 및 세균의 오염이 심하며, 특히 토양 속에서 장기간 생육한 장뇌삼과 산삼의 경우 이러한 현상은 더욱 심하게 나타났기 때문에 다양한 방법으로 소독을 실시하였다.Ginseng to be cultured was used ginseng, camphor ginseng and wild ginseng certified by Yeongju Agricultural Technology Center, Simmani Association and Korean Ginseng Association.When the surface sterilization of the tissue to be cultured, if the concentration of disinfectant solution is high, the tissue is destroyed and disinfectant solution is used. When the concentration of is low, various fungi and bacteria are contaminated. In particular, long-term growth of camphor and wild ginseng has been more severe.

본 발명에서는 사용한 잎과 줄기의 경우 표피조직을 어느 정도 파괴시키면서 내피 조직은 온전히 보호할 수 있는 소독 방법으로써 각각 1-4% 의 고농도 차아염소산 용액과 과산화수소수를 동시에 처리하는 방법을 사용하였다. 일차 소독이 끝난 재료의 표면에는 여전히 소독약제가 묻어있어 시간이 지나면 표면의 소독약제가 다시 내피내의 세포로 침투하여, 잎의 경우 엽육세포가, 줄기의 경우 형성층 조직이 파괴되는 것을 알 수 있었으며, 본 발명의 경우 소독약제를 강하게 처리하기 때문에 멸균수로 2∼3차례 씻어주어도 남은 약제가 천천히 내피 속으로 침투되어 배양 초기에 조직을 죽여버리는 현상이 관찰되어, 이를 극복하기 위하여 표면소독이 끝난 조직을 멸균수에 2∼3번 씻어준 다음, 깨끗한 멸균수에 약 30분 정도 침지시킨 다음 배양 재료로 사용하였다.In the present invention, in the case of the leaves and stems used, the endothelial tissue was completely protected while the epidermal tissue was partially destroyed, and a method of simultaneously treating 1-4% high concentration of hypochlorous acid solution and hydrogen peroxide solution was used. The disinfectant was still on the surface of the first disinfected material, and as time passed, the disinfectant on the surface again penetrated into the cells of the endothelium, whereby the foliar cells in the leaves and the cambium tissue in the stems were destroyed. In this case, since the disinfectant is treated strongly, the remaining agent slowly penetrates into the endothelium and kills the tissue at the beginning of the culture even if it is washed two or three times with sterile water. After washing 2-3 times in water, it was immersed in clean sterile water for about 30 minutes and used as a culture material.

표면소독이 끝난 조직은 영양액이 함유되어 있는 고형배지에 치상한 다음 약 6주간 배양하였을 때, 배양조직으로부터 캘러스와 부정근을 유도할 수 있었다. 이때 배양배지에는 식물 호르몬을 전혀 넣어주지 않는 대신 재료로 사용된 조직을 설탕 3%와 식물생장조절물질인 NAA 및 IBA가 단독 또는 혼합하여 소량(각각 1-10ppm 정도) 함유된 증류수에 일정시간 침적해 둔 다음 배양을 실시하였다. 배양체는 암조건하에서 약 18-26℃, 더욱 바람직하게는 22℃로 조절된 항온실에서 유지시켰다. 조직에서 유도된 캘러스나 부정근은 잘게 썰은 후 액체 배양배지에 넣은 후 진탕배양기에서 약 40∼140 rpm, 더욱 바람직하게는, 60∼100 rpm으로 흔들어 줌으로써 지속적인 생장을 이룰 수 있었다.After disinfection, the tissues were infiltrated in nutrient-containing solid medium and cultured for about 6 weeks, inducing callus and abscesses from the cultured tissues. In this case, do not put any plant hormones in the culture medium, but instead, the tissue used as a material is immersed in distilled water containing a small amount (about 1-10 ppm each) of sugar 3% and plant growth regulators NAA and IBA alone or mixed. It was then cultured. Cultures were maintained in a thermostat controlled to about 18-26 ° C., more preferably 22 ° C. under dark conditions. Callus or intestinal root derived from the tissue was chopped and placed in a liquid culture medium and shaken at about 40 to 140 rpm, more preferably, 60 to 100 rpm in a shaker culture to achieve continuous growth.

식물은 자체적으로 생식 및 생장에 관여하는 식물 호르몬을 지니고 있는데, 배양하려는 잎과 줄기의 형성층 조직은 뿌리와 같은 완전한 양분의 이동 통로 조직을 가지고 있지 않아 고체 배양액에 식물호르몬을 넣어주더라도 배양액 속의 식물호르몬이 배양체 조직으로 용이하게 침투되지 않으며, 배양 초기에 배양조직이 충분한 식물생장조절물질을 배양액으로부터 공급받지 못할 경우 조직이 괴사하는 경우가 인삼류에서는 특히 심하게 나타났다. 이러한 문제점을 극복하기 위해서, 본 발명에서는 배양하고자하는 조직을 삼투조절을 목적으로 하는 설탕 3%와 식물생장조절물질이 함유되어 있는 액체 배양액에 장시간, 바람직하게는 약 48시간 침적해 준 다음 멸균수로 헹구지 않고, 식물생장조절물질이 함유되지 않은 일반 조직배양배지에 접종하여 배양하였다.The plant itself has plant hormones that are involved in reproduction and growth. The stratum corneum of the leaves and stems to be cultured does not have a complete nutrient pathway as roots, so even if plant hormones are added to the solid culture, Hormones do not penetrate easily into culture tissues, and tissues necrosis was especially severe in ginseng if the culture tissues did not receive enough plant growth regulators from the culture medium at the beginning of the culture. In order to overcome this problem, in the present invention, the tissue to be cultured is immersed in a liquid culture solution containing 3% sugar and plant growth regulator for the purpose of osmotic control for a long time, preferably about 48 hours, and then sterilized water. Rinsing was inoculated and cultured by inoculation into general tissue culture medium containing no plant growth regulator.

배양된 조직에서 캘러스와 부정근을 유도하기 위해서 일정주기의 빛을 조사할 수도 있지만, 본 발명에서는 24시간 암상태로 유지시켰으며, 배양 온도 역시 인삼류의 형태형성 반응뿐만 아니라 유도된 캘러스와 부정근의 생장에 결정적인 역할을 한다는 기초 연구결과를 토대로 다양한 온도 조건으로 실험을 실시하였다.Although it may be irradiated with a certain period of light in order to induce callus and inferior root in cultured tissues, in the present invention was maintained in a dark state for 24 hours, the culture temperature also induced the growth of induced callus and inferior root as well as the morphological response of ginseng The experiments were conducted under various temperature conditions based on the results of basic research.

배양 6주 후에 배양조직으로부터 캘러스와 부정근이 유도되는 경우 일반적으로는 유도된 부분을 새로운 배양배지에 옮겨주는 방법을 사용하고 있지만, 본 발명에서는 배양조직이나 배양조직에서 유도된 캘러스 및 부정근이 효율적인 통도조직(물관과 체관)을 만들지 못한 불완전한 상태인 점을 감안하여 배양조직 전체를 새로운 배지에 옮긴 결과 캘러스와 부정근의 생장이 아주 빨라지는 것을 관찰 할 수 있었다.In the case where callus and adventitious roots are induced from cultured tissue after 6 weeks of culture, the method of transferring callus induced part to new culture medium is generally used. However, in the present invention, callus and adventitious roots derived from cultured or cultured tissues are effectively passed. Considering the incomplete status of the tissues (water tube and phloem), the entire culture was transferred to a new medium.

이렇게 하여 얻어진 캘러스와 부정근은 각각 잘게 썰어서 액체배양 배지에 넣어준 다음 진탕배양기에 다양한 속도로 교반하면서 증식을 유도하였는데, 인삼류의 경우 캘러스와 부정근을 잘게 썰어서 배양하는 것이 세포나 부정근의 생체중량 증가에는 크게 효과가 있는 반면, 잘게 써는 과정에서 부서지거나 파괴된 세포가 죽기 전에 액체배양액 속으로 분비하는 유해물질로 인해 배양중인 다른 세포나 부정근의 생장이 정지하는 경우가 종종 발생하였다. 이러한 문제점들은 배양 초기(1-7일 정도, 더욱 바람직하게는 3일)에 가는 그물망을 이용하여 부서진 세포나 찌꺼기들을 제거하고, 배양 며칠 후 다시 새로운 배양액을 교체해 줌으로써 해결될 수 있었다.The callus and the root muscle obtained in this way were finely chopped and placed in a liquid culture medium, and then stirred at various speeds in a shaker incubator to induce proliferation. While very effective, the growth of other cells in the culture or in the roots of the roots of the cultivation often stopped due to the toxic substances secreted into the liquid culture before the broken or destroyed cells died. These problems could be solved by removing broken cells or debris by using a mesh net in the early stage of culture (about 1-7 days, more preferably 3 days), and replacing the new medium again after several days of culture.

이렇게 얻어진 캘러스유래의 세포와 부정근은 그 이후 일반적인 액체현탁 배양 방법에 의해서도 급속한 생장을 보여 주었다.The callus-derived cells and the root muscles thus obtained showed rapid growth even by the usual liquid suspension culture method.

이하 실시예는 본 발명을 보다 상세히 살명하기 위한 것으로, 본 발명이 이들 실시예에 제한되는 것은 아니다.The following examples are intended to illustrate the present invention in more detail, but the present invention is not limited to these examples.

[실시예 1] 배양조직의 표면소독Example 1 Surface Disinfection of Cultured Tissues

수집된 인삼류(인삼, 장뇌삼 및 산삼)로부터 완전한 형태의 잎과 줄기를 3cm 길이로 자른 다음, 재료를 액체비누를 이용해서 재료의 표면을 씻어준 다음, 흐르는 물에서 약 6시간 씻어주었다. 깨끗하게 씻은 조직은 무균상 내에 준비된 유리병에 넣고 소독액을 처리하였는데, 소독액으로는 차아염소산 용액 단용으로 1%, 2%, 3%, 4% 와 과산화수소수 단용으로 0.5%, 1%, 2%, 4%, 및 차아염소산 용액 각각에 대해 과산화수소수를 0.5%, 1%, 2%, 4% 혼용 처리하여 그 영향을 조사하였다.From the collected ginseng (ginseng, camphor ginseng and wild ginseng), the leaves and stems were cut to 3 cm in length, and the material was washed with a liquid soap and then washed with running water for about 6 hours. The cleansed tissue was placed in a glass bottle prepared in a sterile phase and treated with disinfectant solution. For disinfectant solution, 1%, 2%, 3%, 4% for hypochlorous acid solution and 0.5%, 1%, 2% for hydrogen peroxide solution, The effects of 0.5%, 1%, 2%, and 4% of hydrogen peroxide solution were investigated for 4% and hypochlorous acid solutions, respectively.

[실시예 2] 외부 식물생장조절물질의 배양조직 내 전이Example 2 Transfer of External Plant Growth Regulators into Culture Tissue

소독이 끝난 재료 중 잎의 경우는 가로×세로가 약 5mm×5mm 크기로 조제하고, 줄기의 경우 내피를 벗긴 후 다소 목질화 된 내부 조직으로부터 형성층을 분리시킨 다음, 형성층의 길이가 약 1cm 정도가 되도록 조제하여 사용하였다. 배양재료의 내부 세포에 식물생장조절물질을 용이하게 침투시키기 위해 액체 배양액을 이용하였는데, 액체배양액은 내부표준(internal standard)으로써 식물생장조절물질이 처리되지 않은 용액과, NAA와 IBA가 각각 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm, 10ppm 함유된 용액을 이용하였는데, 이때 조직 내 세포의 삼투농도를 안정적으로 해주기 위해 각각의 액체 배양액에 3%의 설탕을 넣어 주었다. 배양재료는 전술한 액체배양액에 12시간, 24시간, 48시간, 72시간 침적 후 식물생장조절물질이 함유되지 않은 고체 배양배지에 접종하였다. 외부표준(external standard)으로써 액체배양액을 처리하지 않은 재료를 0.75% 한천과 동일한 농도의식물생장조절물질이 함유된 고체 배지에 접종하였다.In the case of leaves, the leaves are prepared in the size of about 5mm × 5mm in width and in the case of leaves, and in the case of stems, the inner layers are peeled off, and the forming layers are separated from the somewhat woody internal tissue.The length of the forming layers is about 1cm. It was prepared and used. The liquid culture medium was used to easily infiltrate the plant growth regulator into the internal cells of the culture material. The liquid culture medium is an internal standard, and the solution of the plant growth regulator is not treated, and the NAA and IBA are 1 ppm, respectively. A solution containing 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm, 10ppm was used. At this time, 3% sugar was added to each liquid culture to stabilize the osmolarity of cells in the tissue. The culture material was inoculated into a solid culture medium containing no plant growth regulator after 12 hours, 24 hours, 48 hours, 72 hours of immersion in the liquid culture solution described above. Materials not treated with liquid culture as an external standard were inoculated into a solid medium containing plant growth regulators at the same concentration as 0.75% agar.

[실시예 3] 계대배양 방법 및 배양 온도Example 3 Subculture Method and Culture Temperature

배양 6주 후에 배양조직으로부터 캘러스와 부정근이 유도되면, 1차 계대배양 시에는 배양조직에 새로 유도된 캘러스나 부정근을 그대로 붙여 새로운 배지에 옮겨 주었으며, 2차 계대배양부터는 캘러스와 부정근을 따로 배양하였다. 1차 계대배양 시 캘러스와 부정근을 따로 분리한 후 전술한 방법으로 2차 계대배양한 결과를 대조구로 두어 조사하였다. 인삼류의 세포 및 조직배양을 실시한 기초연구에서 배양온도가 생장에 영향을 줄 수 있다는 결과가 나타남에 따라 배양온도를 18℃에서 26℃까지 각각 1℃ 차이의 범위로 조절하여 그 결과를 관찰하였다.After 6 weeks of incubation, callus and adventitious roots were induced from the cultured tissues, and during the first passage, the newly-induced callus or adventitious roots were attached to the culture medium and transferred to the new medium. . In the first passage, the callus and the root were separated separately, and the results of the second passage in the above-described method were examined as a control. As a result of the basic study of cell and tissue culture of ginseng, it was found that the culture temperature may affect the growth, and the results were observed by adjusting the culture temperature in the range of 1 ℃ difference from 18 ℃ to 26 ℃.

[실시예 4] 배양조직이 분비하는 유해물질의 제거 방법Example 4 Removal of Hazardous Substances Secreted by Cultured Tissues

인삼류의 잎과 줄기 형성층 조직을 이용하여 얻은 캘러스와 부정근을 대량 증식하기 위해서는 액체 현탁배양을 실시하는 것이 바람직하게 여겨지나, 세포를 잘게 썰어서 액체 현탁배양을 할 경우, 썰어주는 과정에서 상처를 입은 세포가 죽어가면서 분비하는 유해 물질로 인하여, 함께 배양한 세포가 생장저해를 받거나 고사하는 문제가 있음으로, 배양 1일, 3일, 7일 후 파괴된 세포나 찌꺼기(debris)를 제거함으로 그 해결책을 얻을 수 있었는데, 제거 방법으로는 서로 다른 농도의 설탕액을 4℃에 24시간 보관한 다음, 서로 섞이지 않게 층을 만들어 세포나 조직의 무게 차에 의해 분리하는 방법과 가는 그물망을 이용하여 분리시키는 방법을 이용하였다.It is preferable to carry out liquid suspension culture in order to massively multiply the callus and intestinal root obtained from the ginseng leaf and stem-forming layer tissues.However, in the case of liquid suspension culture, the wounded cells are cut during the slicing process. Due to the harmful substances secreted by humans, the cells cultured together have a problem of growth inhibition or death, and the solution is removed by removing the destroyed cells or debris after 1, 3 and 7 days of culture. As a method of removal, different concentrations of sugar liquids were stored at 4 ° C for 24 hours, and then layers were formed so as not to mix with each other. Was used.

상기한 바와 같은 실시예에 의하면 인삼류(인삼, 장뇌삼 및 산삼 등)의 잎과 줄기 형성층으로부터 캘러스와 부정근의 유도가 가능하며, 그 증식속도도 상당히 빠른 것으로 나타났다. 인삼류의 잎이나 줄기 조직의 표면소독의 경우 사용된 소독약재 중 차아염소산 용액 2%와 과산화수소수 3%를 혼용 처리시 가장 효과가 우수하였다. 이때 잎의 경우 약 5분, 줄기의 경우 약 10분간 소독함으로써 미생물 오염을 현격히 줄일 수 있었다. 액체배양액을 이용하여 배양액속의 식물생장조절물질을 배양재료로 이동시킨 후 배양하였을 경우 쉽게 캘러스와 부정근이 유도되었으며, 두 재료에서 NAA와 IBA를 각각 3 ppm 농도로 48시간 침적한 것이 가장 효과가 있는 것으로 관찰되었다. 유도된 캘러스와 부정근을 잘게 썰어준 다음 배양 3일째 되는 날 파괴된 세포나 찌꺼기를 가는 그물망을 이용하여 제거시킨 후, 새로운 액체 배지에 계대배양 하였을 경우 쉽게 급속 생장이 가능하였다.According to the embodiment as described above, it is possible to induce callus and intestinal root from the leaf and stem forming layers of ginseng (Ginseng, camphor and wild ginseng), and the growth rate is also very fast. In case of surface disinfection of ginseng leaf or stem tissue, the most effective treatment was 2% hypochlorite solution and 3% hydrogen peroxide solution. At this time, the leaves were sterilized for about 5 minutes and the stems were sterilized for about 10 minutes to reduce microbial contamination. Callus and adventitious roots were easily induced when the plant growth regulator in the culture medium was transferred to the culture material by using liquid culture medium, and it was most effective to incubate 48 hours of NAA and IBA at 3 ppm concentration in both materials. Was observed. Finely cut the induced callus and intestinal roots were removed on the 3rd day of cultivation by removing the destroyed cells or debris using a thin net, and then rapidly grown in a new liquid medium.

본 발명에 의하면 향후 인삼류의 잎이나 줄기조직으로부터 캘러스와 부정근을 대량 증식함으로써, 잎이나 줄기조직에 특징적으로 나타나는 성분을 생산할 수 있으므로 앞으로 새로운 기능성 식품과 의약품 재료 분야에 획기적인 역할을 할 수 있을 것으로 생각된다. 특히 본 발명에 의해서 생산되는 캘러스와 부정근의 경우에 2,4-D를 사용하지 않음으로써 식품으로써의 안정성을 확보할 수 있다는 점에서 그효과가 지극히 크다고 볼 수 있다.According to the present invention, by mass-proliferating callus and adventitious roots from the leaves or stem tissues of ginseng, it is possible to produce components characteristic of the leaves or stem tissues, and thus it may play a significant role in the field of new functional food and pharmaceutical materials in the future. do. In particular, in the case of callus and adventitious root produced by the present invention, the effect is extremely great in that stability as food can be secured by not using 2,4-D.

Claims (11)

인삼류의 잎과 줄기의 형성층을 이용하여 캘러스와 부정근을 유도하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.Callus and adventitious root growth method characterized by inducing callus and adventitious root by using the cambium-forming layer of leaves and stems. 제 1항에 있어서, 인삼류로부터 잎과 줄기 조직을 분리하여 표면 소독을 하는 단계와,The method of claim 1, further comprising the step of surface disinfection by separating leaf and stem tissue from ginseng, 배양액 속에 녹아있는 식물생장조절물질을 상기의 표면 소독된 잎과 줄기 조직으로 침투시키는 단계와,Infiltrating the plant growth regulator dissolved in the culture solution into the surface sterilized leaf and stem tissue; 고체배지에서 상기의 잎과 줄기 조직으로부터 캘러스와 부정근을 유도시키는 단계와,Inducing callus and abscesses from the leaf and stem tissue in a solid medium, 상기 유도된 캘러스와 부정근을 대량증식하는 액체현탁 배양단계를 포함하는 캘러스 및 부정근 증식방법.Callus and adventitious growth method comprising a liquid suspension culture step of mass-proliferating the induced callus and the adventitious muscle. 제 2항에 있어서, 상기의 표면 소독단계는 1- 4% 의 차아염소산 용액과 1-4% 의 과산화수소를 동시에 처리하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.3. The method of claim 2, wherein the surface disinfection step comprises treating 1-4% hypochlorous acid solution and 1-4% hydrogen peroxide simultaneously. 제 2항에 있어서, 상기 침투 단계에서는 상기의 소독된 잎과 줄기를 식물생장조절물질이 함유된 액체배양액에 장시간 침적시키는 것을 특징으로 하는 캘러스및 부정근 증식방법.[Claim 3] The method of claim 2, wherein in the infiltration step, the sterilized leaves and stems are immersed for a long time in a liquid culture solution containing a plant growth regulator. 제 4항에 있어서, 상기의 액체배양액에 설탕을 첨가하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.5. The method of claim 4, wherein sugar is added to the liquid culture solution. 제 4항에 있어서, 상기의 식물생장조절물질로는 NAA와 IBA를 단독 또는 혼합처리하여 액체배양액을 만드는 것을 특징으로 하는 캘러스 및 부정근 증식방법.[Claim 5] The method of claim 4, wherein the plant growth regulator is a method for growing callus and adventitious muscle, characterized in that the liquid culture solution is made by treating NAA and IBA alone or in combination. 제 6항에 있어서, 상기 NAA와 IBA 의 농도는 각각 1-10ppm 정도로 하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.[Claim 7] The method of claim 6, wherein the concentration of NAA and IBA is about 1-10 ppm, respectively. 제 2항에 있어서, 상기 고체배지에서 캘러스와 부정근을 유도시, 조직배양 온도는 18-26℃ 인 것을 특징으로 하는 캘러스 및 부정근 증식방법.[Claim 3] The method of claim 2, wherein, when inducing callus and adventitious root in the solid medium, the tissue culture temperature is 18-26 ° C. 제 2항에 있어서, 상기 고체배지에서 캘러스와 부정근이 유도된 후, 초대배양 시 유도된 캘러스와 부정근이 배양체에 그대로 붙어 있는 상태에서 계대배양 하는 것을 포함함을 특징으로 하는 캘러스 및 부정근 증식방법.3. The method of claim 2, wherein after the callus and the root muscle are induced in the solid medium, the callus and the root muscle induced during primary culture are passaged while being attached to the culture medium. 제 2항에 있어서, 상기의 유도된 캘러스와 부정근의 액체현탁배양 단계는, 유도된 캘러스와 부정근을 잘게 썰어주는 단계와, 배앙 1-5일 후에 단세포나 찌꺼기를 제거한 후 새로운 배양배지에 옮겨주는 단계를 포함하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.According to claim 2, wherein the induced culture of the callus and the root of the roots, the step of slicing the induced callus and the roots of fine, and after 1-5 days of embryos to remove single cells or debris and transfer to a new culture medium Callus and adventitious muscle growth method comprising the step. 제 2항에 있어서, 액체현탁 배양단계시 교반속도는 40 - 140 rpm 으로 하는 것을 특징으로 하는 캘러스 및 부정근 증식방법.3. The method of claim 2, wherein the stirring speed during the liquid suspension culture step is 40-140 rpm.
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