KR102217467B1 - Cultivation method of host plant with plant transformation for plant-based biopharmaceutical manufacturing - Google Patents

Cultivation method of host plant with plant transformation for plant-based biopharmaceutical manufacturing Download PDF

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KR102217467B1
KR102217467B1 KR1020200146580A KR20200146580A KR102217467B1 KR 102217467 B1 KR102217467 B1 KR 102217467B1 KR 1020200146580 A KR1020200146580 A KR 1020200146580A KR 20200146580 A KR20200146580 A KR 20200146580A KR 102217467 B1 KR102217467 B1 KR 102217467B1
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host plant
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temperature
nutrient solution
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박광만
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(주)지플러스생명과학
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A01G31/02—Special apparatus therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
    • A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures

Abstract

A method for cultivating a host plant which has undergone plant transformation in order to prepare a plant-based biomedicine of the present invention comprises: a host plant cultivating step of cultivating a host plant by using a nutrient solution and an artificial light source in a first indoor growing space in which humidity, temperature, and carbon dioxide concentration are adjusted; a gene delivery medium preparation step of injecting a plant expression vector, in which a gene to be produced is combined, in agrobacterium to be delivered to the host plant; an infiltration preparation step of preparing a liquid infiltration containing the agrobacterium with the plant expression vector; an infiltration step of placing a stem part with leaves of the cultivated host plant in an infiltration bath, and making the agrobacterium in the infiltration delivered to the host plant in a condition that a medium with root is expressed to vacuum atmosphere; and a host plant recovery cultivating step of recovering the infiltrated host plant by using the nutrient solution and the artificial light source in a second indoor growing space in which humidity, temperature, and carbon dioxide concentration are adjusted.

Description

식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법{Cultivation method of host plant with plant transformation for plant-based biopharmaceutical manufacturing}Cultivation method of host plant with plant transformation for plant-based biopharmaceutical manufacturing}

본 발명은 바이오 의약품을 생산하기 위한 기주식물의 재배방법에 관한 것으로, 더 상세하게는 기주식물의 내 외래단백질의 일시적 발현을 유도하기 위해 단백질을 암호화하는 유전자로 형질전환 된 아그로박테리움을 기주식물 조직에 침윤시킨 후 재배하는 식물기반 바이오 의약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법에 관한 것이다.The present invention relates to a method for cultivating a host plant for producing a biopharmaceutical, and more particularly, a host plant using Agrobacterium transformed with a gene encoding a protein in order to induce the transient expression of an internal foreign protein of the host plant. It relates to a method for cultivating a host plant that has undergone plant transformation for the production of plant-based biopharmaceuticals grown after infiltrating tissue.

식물 기반 바이오 의약품 제조를 위한 식물을 재배하기 위해서는 재배 환경을 인위적으로 조절하고, 세균, 바이러스, 곰팡이 등에 의한 외부 감염이나 병해충의 침입을 방지하기 위하여 밀폐형 식물 공장 방식이 사용되고 있다. 식물 기반 바이오 의약품에 관련된 단백질 물질을 생산하기 위한 식물은 식물 생장과 함께 단백질물질이 생산될 수 있도록, 식물 재배공정 전에 식물의 형질을 전환하는 과정을 거친다. In order to cultivate plants for the manufacture of plant-based biopharmaceuticals, a closed plant factory method is used to artificially control the cultivation environment and to prevent external infection or invasion of pests by bacteria, viruses, and fungi. Plants for producing protein substances related to plant-based biopharmaceuticals undergo a process of transforming plant traits before plant cultivation so that protein substances can be produced along with plant growth.

한편, 아그로박테리움은 식물에 자신의 DNA를 넣어 증식에 필요한 물질을 만들도록 유도하는데, 연구자들은 이를 이용하여 식물에 발현시키려는 유전자를 아그로박테리움의 DNA에 끼어 넣는다. 숙주(이하 '기주식물'이라 함)에 끼어 들어가는 아그로박테리움의 유전자는 T-DNA(Transfer DNA)라고 하는데, 기주식물이 아크로박테리움에 감염되면서 발현시키려는 유전자가 기주식물 내로 전달되는 과정을 형질전환(transformation)이라 한다. On the other hand, Agrobacterium puts its own DNA in plants and induces them to make substances necessary for proliferation, and researchers use this to insert genes to be expressed in plants into the DNA of Agrobacterium. The gene of Agrobacterium that intervenes in the host (hereinafter referred to as'host plant') is called T-DNA (Transfer DNA). It is a trait that the gene to be expressed is transferred into the host plant when the host plant is infected with Acrobacterium. It is called transformation.

아그로박테리움에 의한 형질전환법은 일반적으로 효율이 높고, 도입되는 유전자의 복제수가 적으며, T-DNA라는 특정의 영역을 단편화시키지 않고 도입할 수 있고, 단시간의 배양에 의해 형질전환체를 얻을 수 있기 때문에 배양변이가 적은 많은 우수한 특징을 갖고 있다. 이 때문에, 여러 가지의 식물종에서 가장 유용한 형질전환의 수법으로서 널리 사용되고 있다.The transformation method with Agrobacterium is generally highly efficient, has a small number of copies of the introduced gene, and can be introduced without fragmenting a specific region called T-DNA, and a transformant can be obtained by culturing for a short time. Because it can be cultured, it has many excellent features with little variation in culture. For this reason, it is widely used as the most useful transformation method in various plant species.

한편, 식물 기반 바이오 의약품 제조를 위해, 아그로박테리움에 투입되는 상기 발현시키고자 하는 유전자는 기주식물에서 대량 생산될 수 있도록 세포 내로 전달되는 DNA 단편(들), 즉 핵산 분자를 지칭하는 벡터와 조합된다. 상기 벡터는 발현시키고자 하는 유전자를 복제시키고 숙주세포에서 독립적으로 재생산될 수 있는 것으로 "전달체"와 호환하여 사용된다. On the other hand, for the production of plant-based biopharmaceuticals, the gene to be expressed that is introduced into Agrobacterium is combined with a DNA fragment(s) delivered into cells, that is, a vector indicating a nucleic acid molecule, so that it can be mass-produced in a host plant. do. The vector is used interchangeably with the "transmitter" to be able to replicate a gene to be expressed and reproduce independently in a host cell.

이와 같이, 식물로부터 원하는 단백질을 대량으로 얻기 위해서 특정 단백질을 암호화(code)하는 유전자가 클로닝 된 플라스미드(plasmid) 혹은 바이러스 벡터(viral vector)를 식물 발현벡터(expression plasmid)라 한다. 즉, 식물 발현벡터는 목적한 유전자의 코딩 서열과, 특정 기주식물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. Ti-플라스미드 벡터는 아그로박테리움 투머파시엔스와 같은 적당한 매개체가 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 식물발현벡터 중 하나이다. 아그로박테리움으로 기주식물을 형질전환시키는 방법에는 크게 두 가지 방식으로 나누어볼 수 있다. 첫번째방법은 아그로박테리움을 액체 배지에 배양하고 꽃을 포함한 지상부, 즉 줄기부를 통째로 담그는 방법이다. 이 방법은 기주식물의 줄기부를 모두 담그기 때문에 아그로박테리움 배양액이 다량으로 필요하다. In this way, in order to obtain a desired protein from plants in large quantities, a plasmid or a viral vector in which a gene encoding a specific protein is cloned is referred to as a plant expression plasmid. That is, the plant expression vector refers to a recombinant DNA molecule comprising a coding sequence of a gene of interest and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host plant. Ti-plasmid vector is one of the plant expression vectors capable of transferring a part of itself, the so-called T-region, to plant cells when a suitable medium such as Agrobacterium tumerfaciens is present. The method of transforming a host plant with Agrobacterium can be divided into two main methods. The first method is to cultivate Agrobacterium in a liquid medium and immerse the above-ground part including flowers, that is, the stem part. This method requires a large amount of Agrobacterium culture medium because it immerses all the stems of the host plant.

두 번째 방법은 아그로박테리움 용액을 농축시킨 후 포말을 만들어 꽃에 묻혀주는 방법이다. 식물의 형질을 전환할 수 있는 또 다른 방법으로 원형질체에 외부 DNA를 주입한 후에 독성 화학 물질에 대한 내성을 바탕으로 DNA가 삽입된 것을 선별하여 캘러스를 만드는 조직 배양(tissue culture)이 있다.The second method is to make a foam after concentrating the Agrobacterium solution and bury it on the flower. Another way to transform a plant's trait is tissue culture, in which foreign DNA is injected into a protoplast and then a callus is made by selecting the inserted DNA based on resistance to toxic chemicals.

대한민국 공개특허 제10-2002-0092915호와 제10-2002-0092916호에는 각각 식물세포를 원심분리처리 및 열처리와 원심분리 처리하는 방법과 같은 전처리에 의해 유전자도입 효율을 향상시키는 방법이 게시되어 있다. 또한, 한국 공개특허 제10-2004-0086974호에는 당 및 염 전처리 과정을 통한 아그로박테리움에 의한 식물체 형질전환 효율을 증진시키는 방법이 기재되어 있다.Korean Patent Laid-Open Nos. 10-2002-0092915 and 10-2002-0092916 disclose methods of improving transgenic efficiency through pretreatment such as centrifugation treatment, heat treatment, and centrifugation treatment of plant cells, respectively. . In addition, Korean Patent Application Laid-Open No. 10-2004-0086974 describes a method of enhancing plant transformation efficiency by Agrobacterium through sugar and salt pretreatment.

한편, 상술한 바와 같이 형질전환이 이루어진 기주식물 재배시설에 의해 대량으로 재배되고, 기주식물로부터 바이오 의약품을 추출하게 되는데, 상기와 같이 기주식물의 재배는 안정적인 공급을 위하여 특정한 식물 재배시설에서 재배된다. On the other hand, as described above, it is cultivated in large quantities by the host plant cultivation facility where the transformation has been performed, and biopharmaceuticals are extracted from the host plant. As described above, the cultivation of the host plant is cultivated in a specific plant cultivation facility for stable supply. .

본 출원인은 대한민국 등록특허 제 10-1933986호에 밀폐형 식물 공장의 분진제어 시스템 및 이의 제어방법과, 대한민국 등록특허 제 10-1982817호에 패키지형 항온항습기 방식 밀폐형 식물공장의 환경제어시스템 및 이의 제어방법, 대한민국 특허등록 제 10-1931750호에 공조기 방식 밀폐형 식물공장의 환경제어 시스템 및 이의 제어방법을 등록 받은 바 있다. The applicant of the present invention is a dust control system and a control method of a closed plant factory in Korean Patent Registration No. 10-1933986, and an environmental control system and a control method thereof in a packaged thermo-hygrostat method in Korean Patent No. 10-1982817. , In Korea Patent Registration No. 10-1931750, the environmental control system and its control method of an air conditioner-type enclosed plant factory have been registered.

일본 공개 특허 제 1995호에는 환경제어 입체식 식물공장이 게시되어 있으며, 일본 공개특허 제 2013-005772호에는 식물육성공간 제어시스템이 게시되어 있다. Japanese Laid-Open Patent No. 1995 discloses a three-dimensional plant factory for environmental control, and Japanese Laid-Open Patent No. 2013-005772 discloses a plant breeding space control system.

대한민국 특허등록 10-1819585호에는 파장변경이 가능한 엘이디 조명장치를 이용한 식물재배기가 게시되어 있으며, 일본특허 공개 제 2019-518961호에는 LED광을 이용한 식물을 재배하는 방법 및 이를 이용한 LED광 시스템이 게시되어 있다. 일본특허등록 제 6594970호에는 식물의 초기 생장효율을 최적화 하는 LED 조명모듈이 게시되어 있으며, 일본 공개특허 제 2019-106955호에는 식물의 재배방법이 게시되어 있다. Korean Patent Registration No. 10-1819585 discloses a plant cultivation machine using an LED lighting device capable of changing wavelength, and Japanese Patent Publication No. 2019-518961 discloses a method of cultivating plants using LED light and an LED light system using the same. Has been. Japanese Patent Registration No. 6594970 discloses an LED lighting module that optimizes the initial growth efficiency of plants, and Japanese Patent Laid-Open No. 2019-106955 discloses a plant cultivation method.

상술한 바와 같이 발광다이오드와 같은 인조광원을 이용하여 식물을 재배하는 재배장치는 인조광원으로부터 조사되는 광량, 암기시간, 파장대 등을 한정하고 있으나 식물의 형질 전환이 이루어진 기주식물의 재배를 위한 많은 제약을 충족시킬 수 없다.As described above, the cultivation apparatus for cultivating plants using an artificial light source such as a light-emitting diode limits the amount of light irradiated from the artificial light source, the memorization time, and the wavelength range, but there are many restrictions for the cultivation of the host plant in which the plant has been transformed. Can't meet

대한민국 공개특허 제 제10-2002-0092915호Republic of Korea Patent Publication No. 10-2002-0092915 대한민국 등록특허 제 10-1933986호Korean Patent Registration No. 10-1933986 일본 공개 특허 제 1995호Japanese Published Patent No. 1995 일본 공개특허 제 2019-106955호Japanese Patent Laid-Open No. 2019-106955 일본 공개특허 제 2013-005772호Japanese Patent Application Publication No. 2013-005772

본 발명은 상술한 종래의 문제점을 감안하여 기주식물의 재배와 식물 형질 전환이 이루어진 기주식물의 회복을 위한 양호한 생육조건을 제공하여 대량의 기주식물의 생산이 가능한 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법을 제공함에 있다.The present invention provides good growth conditions for the cultivation of the host plant and the recovery of the host plant that has undergone plant transformation in view of the above-described conventional problems, and thus, a plant trait for manufacturing a plant-based biopharmaceutical capable of producing a large amount of host plants. It is to provide a method of cultivating host plants that have been converted.

상기와 같은 목적을 달성하기 위한 본 발명의 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법은 습도와 온도 및 이산화탄소의 농도가 조절된 제1 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 재배하는 기주식물 재배단계;In order to achieve the above object, the method of cultivating a host plant in which plant transformation has been performed for the production of plant-based biopharmaceuticals of the present invention comprises nutrient solutions and artificial light sources in a first indoor growth space in which humidity, temperature, and carbon dioxide concentration are controlled. A host plant cultivation step of cultivating a host plant by using;

생산하고자 하는 유전자가 조합된 식물발현벡터를 기주식물로 운반할 아그로박테리움에 주입하는 유전자 운반 매개체 준비단계와, 상기 식물발현벡터가 포함된 상기 아그로박테리움을 함유하는 액상의 침윤액을 준비하는 침윤액 준비단계와;Preparing a gene delivery medium in which a plant expression vector in which the gene to be produced is combined is injected into Agrobacterium to be transported to a host plant, and a liquid infiltrate containing the Agrobacterium containing the plant expression vector is prepared. Preparing the infiltrate;

재배된 기주식물의 잎을 포함한 줄기부를 상기 침윤조에 담그고, 뿌리를 포함한 배지가 진공분위기에 노출된 상태에서 침윤액 내의 상기 아그로박테리움이 상기 기주식물로 전달될 수 있도록 하는 침윤단계와; An infiltration step of immersing the stem portion including the leaves of the cultivated host plant in the infiltration tank, and allowing the Agrobacterium in the infiltrate to be transferred to the host plant while the medium including the roots is exposed to the vacuum atmosphere;

상기 침윤이 이루어진 기주식물을 습도와 온도 및 이산화탄소의 농도가 조절된 제2 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 회복시키는 기주식물회복재배단계를 포함한다. And a host plant recovery cultivation step of recovering the host plant by using a nutrient solution and an artificial light source in a second indoor growth space in which the infiltrated host plant is controlled in humidity, temperature, and carbon dioxide concentration.

본 발명에 있어서, 상기 기주식물은 니벤타이며, 제1,2실내생육공간 내의 상기 이산화탄소의 농도는 350 내지 400ppm 이다.In the present invention, the host plant is niventa, and the concentration of the carbon dioxide in the first and second indoor growth spaces is 350 to 400 ppm.

그리고 상기 침윤단계에 있어서, 상기 기주식물을 상기 침윤액에 침지시키기 전 진공분위기에 기주식물을 노출시키는 기주식물진공노출단계를 더 구비한다.And in the infiltration step, further comprising a host plant vacuum exposure step of exposing the host plant to a vacuum atmosphere before immersing the host plant in the infiltration liquid.

상기 기주식물재배단계와 기주식물회복재배단계에 있어서, 제1,2실내생육공간의 조절된 온도을 온도센서를 이용하여 제1온도검출단계와, 재배판의 배지에 공급되는 양액의 온도를 검출하는 제 2온도검출단계와, 상기 제 1,2온도검출단계에서 검출된 온도를 비교하여 상기 양액의 온도를 제1,2생육공간 내의 온도보다 낮추는 양액온도 조절단계를 더 구비한다. In the host plant cultivation step and the host plant recovery cultivation step, a first temperature detection step using a temperature sensor to control the temperature of the first and second indoor growth spaces, and detecting the temperature of the nutrient solution supplied to the medium of the cultivation plate. A second temperature detection step and a nutrient solution temperature control step of lowering the temperature of the nutrient solution than the temperature in the first and second growth spaces by comparing the temperature detected in the first and second temperature detection steps.

본 발명에 따른 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법은 기주식물인 니벤타를 이용하여 바이오 의약품을 만들 수 있도록 유전자를 주입하고, 재배시설을 이용하여 단기간 내에 대량 재배생산 할 수 있으므로 산업적으로 유용한 고부가가치의 단백질 생산을 가능하게 한다. The cultivation method of a host plant that has undergone plant transformation for the production of plant-based biopharmaceuticals according to the present invention is to inject a gene to make biopharmaceuticals using the host plant niventa, and cultivate mass in a short period using a cultivation facility. As it can be produced, it enables the production of high value-added proteins that are industrially useful.

도 1은 본 발명의 따른 식물기반 바이오의약품 제조를 위한 식물 형질 전환이 이루어진 기주식물의 재배방법을 나타내 보인 블록도. 1 is a block diagram showing a cultivation method of a host plant in which plant transformation has been made for manufacturing a plant-based biopharmaceutical according to the present invention.

본 발명에 따른 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법의 일 실시예를 도 1에 나타내 보였다. Fig. 1 shows an example of a method for cultivating a host plant in which plant transformation has been performed for manufacturing a plant-based biopharmaceutical according to the present invention.

도면을 참조하면, 본 발명에 따른 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법(100)은 습도와 온도 및 이산화탄소의 농도가 조절된 제1실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 재배하는 기주식물 재배단계(110)를 포함한다. Referring to the drawings, the cultivation method 100 of the host plant in which the plant transformation has been made for the production of plant-based biopharmaceuticals according to the present invention is a nutrient solution and an artificial light source in a first indoor growth space in which humidity, temperature and carbon dioxide concentration are controlled. It includes a host plant cultivation step 110 of cultivating the host plant by using.

그리고 생산하고자 하는 유전자가 조합된 식물발현벡터를 기주식물로 운반할 아그로박테리움에 주입하는 유전자 운반 매개체 준비단계(120)와, 상기 식물발현벡터가 포함된 상기 아그로박테리움을 함유하는 액상의 침윤액을 밀폐 가능한 챔버 내에 마련된 침윤조에 공급하는 침윤액 준비단계(130)를 구비한다. And the gene transport medium preparation step 120 of injecting the plant expression vector in which the gene to be produced is combined into the Agrobacterium to be transported to the host plant, and the infiltration of a liquid containing the Agrobacterium containing the plant expression vector. It includes an infiltration liquid preparation step 130 for supplying the liquid to the infiltration tank provided in the sealable chamber.

그리고 상기 기주식물재배단계(110)에 의해 재배된 기주식물의 잎을 포함한 줄기부를 상기 침윤조의 침윤액에 담그고, 포트에 지지된 배지 포함하는 뿌리부가 진공분위기에 노출된 상태에서 침윤액 내의 상기 아그로박테리움이 상기 기주식물로 전달될 수 있도록 하는 침윤단계(140)와, 침윤이 이루어진 기주식물을 습도와 온도 및 이산화탄소의 농도가 조절된 제2 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 회복시키는 기주식물회복재배단계(170)를 포함한다.And the stem part including the leaf of the host plant cultivated by the host plant cultivation step 110 is immersed in the infiltration liquid of the infiltration tank, and the agro in the infiltration liquid in a state where the root part including the medium supported by the pot is exposed to a vacuum atmosphere. Infiltrating step 140 to allow bacterium to be transferred to the host plant, and host plant using nutrient solution and artificial light source in a second indoor growth space in which humidity, temperature, and concentration of carbon dioxide are controlled by infiltrating the host plant It includes a host plant recovery cultivation step 170 to recover.

상술한 바와 같이 구성된 본 발명에 따른 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 제배방법을 단계별로 보다 상세하게 설명하면 다음과 같다.The method for cultivating the host plant in which the plant transformation has been performed for the production of plant-based biopharmaceuticals according to the present invention constructed as described above will be described in more detail step by step as follows.

먼저, 상기 기주식물재배단계(110)에 있어서, 기주식물의 재배는 건축물구조물에는 내부에 마련된 제1실내생육공간에서 이루어진다. First, in the host plant cultivation step 110, the host plant is cultivated in a first indoor growth space provided inside the building structure.

상기 건축구조물에는 제1실내생육공간이 지주식물을 재배하기 위한 최상의 상태로 유지할 수 있도록 양액공급부를 포함하는 양액공급유닛, 항온항습유닛 및 이산화탄소공급유닛, 분진제어유닛 등이 설치되는데, 이러한 기술적 구성은 본원 발명인이 특허출원하여 등록 받은 대한민국 등록특허 제 10-1933986호에 밀폐형 식물 공장의 분진제어 시스템 및 이의 제어방법과, 대한민국 등록특허 제 10-1982817호에 패키지형 항온항습기 방식 밀폐형 식물공장의 환경제어시스템 및 이의 제어방법, 대한민국 특허등록 제 10-1931750호에 공조기 방식 밀폐형 식물공장의 환경제어 시스템 및 이의 제어방법에 상세하기 기재되어 있으므로 다시 설명하지 않기로 한다. In the building structure, a nutrient solution supply unit including a nutrient solution supply unit, a constant temperature and humidity unit, a carbon dioxide supply unit, a dust control unit, etc. are installed so that the first indoor growth space can be maintained in the best condition for cultivating the holding plant. The dust control system and its control method of a closed plant factory in Republic of Korea Patent Registration No. 10-1933986 registered by the inventor of the present invention, and the environment of a package-type thermo-hygrostat method sealed plant factory in Korea Patent No. 10-1982817 The control system and its control method, Korean Patent Registration No. 10-1931750, describes in detail the environmental control system and its control method of an air conditioner-type sealed plant factory, so it will not be described again.

상기 기주식물재배단계(110)에 있어서, 상기 제1실내생육공간 내에서 바이오약품의 생산을 위한 기주식물로서 니벤타가 재배될 수 있다. In the host plant cultivation step 110, niventa may be cultivated as a host plant for the production of biopharmaceuticals in the first indoor growth space.

상기 기주식물은 제1실내생육공간 내에서 모래, 점토 비드들(clay beads), 폴리스티렌 접시들 또는 암면(rock wool)과 같은 중성의 배지가 설치되는 트레이를 이용하여 양액재배될 수 있다. 상기 배지는 기주식물에 무기물 염과 필수 영양소들을 공급하는 양액 스트림으로 양액공급유닛에 의해 규칙적으로 관개된다.The host plant may be cultivated in a nutrient solution using a tray in which a neutral medium such as sand, clay beads, polystyrene dishes or rock wool is installed in the first indoor growth space. The medium is a nutrient solution stream that supplies mineral salts and essential nutrients to the host plant and is regularly irrigated by the nutrient solution supply unit.

한편, 기주식물이 재배되는 제1 실내생육공간 내의 습도는 기주식물의 발이시기와 재배시기에 다르게 조절될 수 있다. 상기 발아단계의 습도는 55% 내지 65%를 유지하며, 재배시기의 습도는 50% 내지 60%를 유지함이 바람직하다. On the other hand, the humidity in the first indoor growth space in which the host plant is cultivated may be adjusted differently depending on the time when the host plant is grown and when the host plant is grown. The humidity of the germination step is maintained at 55% to 65%, and the humidity at the cultivation period is preferably maintained at 50% to 60%.

그리고 항온항습유닛을 이용한 제 1실내생육공간의 온도는 하절기와 동절기를 다르게 적용할 수 있는데, 하절기는 20 도 내지 26도를 유지하고, 동절기에는 22도 내지 28도를 유지함이 바람직하다. 그러나 상기 제 1실내생육공간의 온도는 상술한 실시예에 의해 한정되지 않고, 습도와, 제1실내생육공간 내부에서의 기류흐름에 따라 달라질 수 있다. 예컨대, 실내의 온도의 조절은 인조광원을 발광다이오드를 이용하는 경우, 발광다이오드로부터 발생되는 열을 감암하여 조정함이 바람직하다. In addition, the temperature of the first indoor growth space using the constant temperature and humidity unit can be applied differently in summer and winter, and it is preferable to maintain 20 to 26 degrees in summer and 22 to 28 degrees in winter. However, the temperature of the first indoor growth space is not limited by the above-described embodiment, and may vary depending on humidity and airflow in the first indoor growth space. For example, when a light emitting diode is used as an artificial light source, the indoor temperature is preferably adjusted by sensitizing heat generated from the light emitting diode.

한편, 상기 제1실내생육공간의 내의 이산화탄소의 농도는 390ppm 내지 420ppm을 유지함이 바람직하다. 엽체류인 기주식물을 재배함에 있어서, 390ppm 이하의 경우 발육이 지연되는 경향이 있었으며, 내지 420ppm의 이상인 경우, 이의 경계점으로부터 성장된 잎의 폭이 설정된 농도에서 자란 잎에 비하여 평균 3mm 작았으며, 길이는 2.8mm 정도 작게 자라는 것을 알 수 있었다.On the other hand, the concentration of carbon dioxide in the first indoor growth space is preferably maintained at 390ppm to 420ppm. In cultivating the host plant, which is a foliar, growth tended to be delayed when the concentration was less than 390 ppm, and in the case of more than 420 ppm, the width of the leaf grown from the boundary point was 3 mm smaller on average than the leaf grown at the set concentration, and the length Was found to grow as small as 2.8mm.

이의 결과는 이산화탄소의 농도를 390ppm 내지 420ppm로 유지한 제1실내생육공간에서 자란 지주식물의 잎 100장을 무작위로 채취하고, 이산화 탄소의 농도가 423ppm 내지 430ppm가 유지된 생육공간에서 재배한 지주식물의 잎을 부작위로 100장을 채취하여 측정한 평균값이다. 이 실험에서 상기 온도와 재배를 위한 양액 및 기타조건을 상호 동일하게 유지하였으며, 이산화 탄소의 농도는 실질적으로 적용 시 이산화탄소의 농도 조절 및 측정위치에 따라 ±5ppm 이상의 오차가 있었다. The result of this is that 100 leaves of a holding plant grown in the first indoor growth space with a carbon dioxide concentration of 390 ppm to 420 ppm were randomly collected, and a holding plant grown in a growth space with a carbon dioxide concentration of 423 ppm to 430 ppm. It is the average value measured by collecting 100 leaves of the tree as an omission. In this experiment, the temperature, nutrient solution and other conditions for cultivation were kept the same, and the concentration of carbon dioxide actually had an error of ±5 ppm or more depending on the concentration control and measurement location of carbon dioxide when applied.

한편, 상기 기주식물재배단계(110)와 후술하는 기주식물 회복단계는 제1,2실내생육공간의 조절된 온도을 온도센서를 이용하여 검출하는 제1온도검출단계와, 재배판의 배지에 공급되는 양액의 온도를 검출하는 제 2온도검출단계와, 상기 제 1,2온도검출단계에서 검출된 온도를 비교하여 상기 양액의 온도를 제1,2생육공간 내의 온도보다 낮추는 양액온도 조절단계를 각각 더 구비한다. 이는 지주식물의 재배 시 인조광원인 발광다이오드 또는 램프로부터 발생되는 열에 의해 제1,2실내생육공간의 내주온도와 양액의 온도가 실질적으로 유사해질 수 있으므로 지주식물의 뿌리온도를 줄기와 잎에 비해 낮추기 위해 양액의 온도제어가 필요하다. 이는 지주식물의 성장에 영향을 주는 주요한 요소로 작용한다. On the other hand, the host plant cultivation step 110 and the host plant recovery step to be described later include a first temperature detection step of detecting the controlled temperature of the first and second indoor growth spaces using a temperature sensor, and supply to the medium of the cultivation plate. A second temperature detection step of detecting the temperature of the nutrient solution and a nutrient solution temperature control step of lowering the temperature of the nutrient solution below the temperature in the first and second growth spaces by comparing the temperature detected in the first and second temperature detection steps, respectively. Equipped. This is because the inner circumferential temperature of the first and second indoor growth spaces and the temperature of the nutrient solution may become substantially similar due to heat generated from the light-emitting diodes or lamps, which are artificial light sources during cultivation of the prop plant. To lower it, temperature control of the nutrient solution is required. This acts as a major factor affecting the growth of the holding plant.

상기 유전자 운반 매개체 준비단계(120)는 식물기반 바이오의약품 제조를 위해 기주식물을 통해 생산하고자 하는 유전자를 조합한 식물발현벡터를 아그로박테리움에 주입시키는 단계이다. 식물발현벡터는 전기천공법에 의해 아그로박테리움 투메파시엔스와 같은 매개체에 삽입될 수 있다. 식물발현벡터가 삽입되어 형질전환된 아그로박테리움 세포는 제한효소 절단과 전기영동을 통하여 유전자 삽입을 확인할 수 있다.The preparation step 120 of the gene transport medium is a step of injecting a plant expression vector in which a gene to be produced through a host plant is combined into Agrobacterium for manufacturing a plant-based biopharmaceutical. Plant expression vectors can be inserted into a vector such as Agrobacterium tumefaciens by electroporation. Agrobacterium cells transformed by inserting a plant expression vector can confirm gene insertion through restriction enzyme digestion and electrophoresis.

특정 유전자가 조합된 식물발현벡터를 구축하는 방법은 공지된 방법이 적용되며 상세한 설명은 생략한다. 상기 특정 유전자는 식물기반 바이오의약품 제조를 위한 것으로서 한정되지 않는다. A known method is applied to a method of constructing a plant expression vector in which a specific gene is combined, and detailed descriptions are omitted. The specific gene is for manufacturing plant-based biopharmaceuticals and is not limited.

그리고, 아그로박테리움에 식물발현벡터를 삽입하는 방법은 공개된 크리스퍼(CRISPR/Cas9), 즉 '유전자 가위'를 이용한 기법이 적용될 수 있다. In addition, as a method of inserting a plant expression vector into Agrobacterium, a technique using the disclosed CRISPR (CRISPR/Cas9), that is,'gene scissors' can be applied.

상기 침윤액 준비단계(130)는 형질전환 된 아그로박테리움을 전배양하여, 10mM MES(2-(N-morpholino) ethanesulfonic acid, pH 55), 10mM MgCl2, 20uM 아세토시린곤(acetosyringone), 50㎍/mL 카나마이신(kanamycin) 및 100㎍/mL 리팜피신(rifampicin)이 들어간 YEP(yeastextract peptone) 배양액에서 28℃로 배양하는 아그로박테리움 배양단계와; 배양액을 MES(pH 56), MgCl 2 및 아세토시린곤이 포함된 MS 기본 배지에 희석하여 침윤액을 조성하는 희석단계를; 포함할 수 있다. 침윤액은 기주식물 침윤 전에 상온에서 2~ 3시간 배양시키는 것이 바람직하다.In the infiltrate preparation step 130, the transformed Agrobacterium is pre-cultured, and 10mM MES (2-(N-morpholino) ethanesulfonic acid, pH 55), 10mM MgCl2, 20uM acetosyringone, 50µg Agrobacterium culture step of culturing at 28° C. in YEP (yeastextract peptone) culture solution containing /mL kanamycin and 100㎍/mL rifampicin ; A dilution step of diluting the culture solution in MS basal medium containing MES (pH 56), MgCl 2 and acetosyringone to form an infiltrate; Can include. It is preferable to incubate the infiltrate at room temperature for 2 to 3 hours before infiltrating the host plant.

상기 침윤단계(140)는 기주식물의 뿌리부가 빈공부위기에 노출되고, 잎과 줄기부가 침윤조에 저장된 침윤액에 잠기게 함으로써 이루어진다. 이때에 상기 뿌리는 상술한 바와 같이 트레이에 설치된 배지에 활작이 이루어진 상태로 진공분위기에 노출된다. 이는 침윤공정이 이루어진 후 기주식물의 회복력을 높이기 위함이다. The infiltrating step 140 is performed by exposing the root portion of the host plant to the void area, and immersing the leaves and stem portions in the infiltrating liquid stored in the infiltration tank. At this time, the roots are exposed to a vacuum atmosphere in a state in which the culture medium installed on the tray has been activated as described above. This is to increase the resilience of the host plant after the infiltration process is performed.

상기 기주식물의 줄기와 잎을 침윤액에 침윤하는 시간은 기주식물에 따라 다르나, 6시간 내지 12시간이 바람직하다.The time for infiltrating the stem and leaves of the host plant into the infiltrate varies depending on the host plant, but is preferably 6 to 12 hours.

상기 침윤단계에 있어서, 상기 기주식물의 잎과 줄기가 침윤액에 잠기기 전에 잎과 줄기로부터 가스가 방출될 수 있도록 진공부위기에 노출시키는 기주식물진공노출단계(150)을 더 구비할 수 있으며, 상기 기주식물이 침윤이 이루어져 침윤액과 분리된 기주식물의 잎과 줄기에 진공이 해지됨으로서 대기압이 가하여지도록 하는 기주식물가압단계(160)을 더 구비할 수 있다. 상기 침윤단계는 진공 및 진공해지가 가능한 친공챔버의 내부에 침윤액이 담긴 침윤조를 설치하고 이를 이용하여 수행함이 바람직하다.In the infiltration step, a host plant vacuum exposure step 150 may be further provided in which the leaves and stems of the host plant are exposed to a vacuum area so that gas can be released from the leaves and stems before being immersed in the infiltrating solution, The host plant pressure step 160 in which atmospheric pressure is applied by infiltrating the host plant and releasing the vacuum on the leaves and stems of the host plant separated from the infiltrate may be further provided. The infiltrating step is preferably performed by installing an infiltrating tank containing the infiltrating liquid in the inside of the pore-pore chamber capable of vacuuming and releasing the vacuum, and using it.

상기 기주식물회복재배단계(170)은 상기 기주식물재배단계(110)와 같이 습도와 온도 및 이산화탄소의 농도가 조절된 제2 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 회복시키는 단계이다. 상기 제2실내생육공간은 상기 기주식물재배단계(110)와 같이 건축구조물에 설치되는데, 이 건축구조물에는 지주식물을 재배하기 위한 최상의 상태로 유지할 수 있도록 앵액공급부를 포함하는 양액공급유닛, 항온항습유닛 및 이산화 탄소공급유닛, 분진제어유닛 등이 설치된다.The host plant recovery cultivation step 170 is a step of restoring the host plant by using a nutrient solution and an artificial light source in a second indoor growth space in which humidity, temperature and carbon dioxide concentration are controlled as in the host plant cultivation step 110. . The second indoor growth space is installed in the building structure as in the host plant cultivation step 110, and the building structure includes a nutrient solution supply unit including a sakura solution supply unit so as to maintain the best condition for cultivating the holding plant, constant temperature and humidity. A unit, a carbon dioxide supply unit, and a dust control unit are installed.

상기 기주식물회복재배단계(170)는 상술한 바와 같이 기주식물의 침윤단계(140)의 완료로 인하여 기주식물의 형질전환이 완료된 기주식물을 제 2실내생육공간의 양액재배시설에 이식되어 재배됨으로써 이루어지는데, 상기 회복을 위한 재배조건은 실질적으로 기주식물 재배단계(110)와 동일하다. In the host plant recovery and cultivation step 170, as described above, the host plant, which has been transformed by the host plant due to the completion of the infiltration step 140, is transplanted and cultivated in the nutrient solution cultivation facility of the second indoor growth space. It is made, the cultivation conditions for the recovery are substantially the same as the host plant cultivation step (110).

상기 기주식물회복재배단계에 있어서, 기주식물을 회복 및 배재하기 위한 양액은 물에 질산칼슘, 질산가리, 질산암모늄, 킬레이트철 등이 포함된 A타입양액과, 질산가리, 제1인산가리, 황산마그네슘, 황산가리, 붕산, 황산망간, 황산아연, 황산구리, 몰리브덴산나트륨 등이 포함된 B타입양액이 선택적으로 사용될 수 있다. In the host plant recovery and cultivation step, the nutrient solution for recovering and excluding the host plant is a type A nutrient solution containing calcium nitrate, nitrate, ammonium nitrate, iron chelating, etc. in water, nitrate, first phosphate, sulfuric acid. A B-type nutrient solution containing magnesium, potassium sulfate, boric acid, manganese sulfate, zinc sulfate, copper sulfate, sodium molybdate, and the like may be optionally used.

상술한 양액을 이용한 기주식물의재배단계(110)와 기주식물의회복재배단계(170)에 있어서 상기 기주식물을 재배하기 위한 양액의 PH 값을 센서를 이용하여 검출하고, 양액의 PH 값이 5.6 내지 6.9를 유지하도록 하는 PH 조절단계를 더 포함할 수 있다. 이는 기주식물이 성장함에 따라 양액의 PH값을 보상한다.In the cultivation step 110 of the host plant using the above-described nutrient solution and the recovery cultivation step 170 of the host plant, the PH value of the nutrient solution for cultivating the host plant is detected using a sensor, and the pH value of the nutrient solution is 5.6 It may further include a PH adjustment step to maintain the to 6.9. This compensates for the pH value of the nutrient solution as the host plant grows.

또한 기주식물재배단계(110)와 기주식물회복재배단계(170)에 있어서, 기주식물에 발광다이오드와 같은 인조광원을 이용하여 광을 조사하는 명기의 시간은 1일 기준 15 내지 17시간을 조사함이 바람직하며, 광을 조하하지 않은 암기 시간은 7시간 내지 9시간을 조사함이 바람직하다. 이때에 조사되는 인조광원으로부터 208±20 ㎛ol/㎡s 의 광량을 조사함이 바람직하다. 그러나 이에 한정되지 않고, 식물 재배기간 생육상태에 따라 명기와 암기의 시간 및 조사되는 광량은 163 내지 235 ㎛ol/㎡s의 범위내에서 조사될 수 있다.In addition, in the host plant cultivation step 110 and the host plant recovery cultivation step 170, the specific time of irradiating the host plant with light using an artificial light source such as a light emitting diode is irradiated for 15 to 17 hours per day. This is preferable, and it is preferable to irradiate 7 hours to 9 hours for the memorization time without dimming the light. At this time, it is preferable to irradiate an amount of light of 208±20 μmol/m²s from the artificial light source to be irradiated. However, the present invention is not limited thereto, and the light intensity and the amount of light irradiated may be irradiated within the range of 163 to 235 ㎛ol/m2s depending on the growing state of the plant cultivation period.

상술한 바와 같이 구성된 본 발명에 따른 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법은 기주식물인 니벤타를 이용하여 바이오 의약품을 만들 수 있도록 유전자를 주입하고, 재배시설을 이용하여 단기간내에 대량 재배생산 할 수 있다. The cultivation method of a host plant in which plant transformation has been carried out for the production of plant-based biopharmaceuticals according to the present invention constituted as described above is to inject a gene to make a biopharmaceutical using the host plant niventa, and use a cultivation facility. Therefore, mass cultivation can be produced within a short period of time.

본 발명은 도면에 도시된 실시예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 본 기술 분야의 통상의 지식을 가진 사람이라면 이로부터 다양한 변형 및 균등한 타 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 등록 청구 범위의 기술적 사상에 의해 정해져야 할 것이다.The present invention has been described with reference to the embodiments shown in the drawings, but these are only exemplary, and those of ordinary skill in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical idea of the attached registration claims.

Claims (7)

습도와 온도 및 이산화탄소의 농도가 조절된 제1 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 재배하는 기주식물 재배단계:
생산하고자 하는 유전자가 조합된 식물발현벡터를 기주식물로 운반할 아그로박테리움에 주입하는 유전자 운반 매개체 준비단계와, 상기 식물발현벡터가 포함된 상기 아그로박테리움을 함유하는 액상의 침윤액을 준비하는 침윤액 준비단계와;
재배된 기주식물의 잎을 포함한 줄기부를 침윤조에 담그고, 뿌리를 포함한 배지가 진공분위기에 노출된 상태에서 침윤액 내의 상기 아그로박테리움이 상기 기주식물로 전달될 수 있도록 하는 침윤단계와;
상기 침윤이 이루어진 기주식물을 습도와 온도 및 이산화탄소의 농도가 조절된 제2 실내생육공간에서 양액과 인조광원을 이용하여 기주식물을 회복시키는 기주식물회복재배단계를 포함하며,
상기 침윤단계는
상기 기주식물을 상기 침윤액에 침지시키기 전 기주식물을 진공분위기에 노출시키는 기주식물진공노출단계와,
상기 기주식물을 상기 침윤액에 침지시키기 후 진공을 해지하여 기주식물에 압력이 작용하도록하는 기주식물가압단계를 구비한 것을 특징으로 하는 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법.The host plant cultivation step of cultivating a host plant using a nutrient solution and an artificial light source in the first indoor growth space in which humidity, temperature and carbon dioxide concentration are controlled:
Preparing a gene delivery medium in which a plant expression vector in which the gene to be produced is combined is injected into Agrobacterium to be transported to a host plant, and a liquid infiltrate containing the Agrobacterium containing the plant expression vector is prepared. Preparing the infiltrate;
An infiltrating step of immersing the stem including the leaves of the cultivated host plant in an infiltration tank, and allowing the Agrobacterium in the infiltrate to be transferred to the host plant while the medium including the roots is exposed to the vacuum atmosphere;
A host plant recovery cultivation step of recovering the host plant by using a nutrient solution and an artificial light source in a second indoor growing space in which the infiltrated host plant is controlled in humidity, temperature, and carbon dioxide concentration,
The infiltration step
A host plant vacuum exposure step of exposing the host plant to a vacuum atmosphere before immersing the host plant in the infiltrate,
Cultivation of host plants in which plant transformation has been performed for the production of plant-based biopharmaceuticals, characterized in that the host plant is pressurized by releasing the vacuum after immersing the host plant in the infiltrating solution to apply pressure to the host plant Way. 제 1항에 있어서,
상기 기주식물은 니벤타인 것을 특징으로 하는 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법.The method of claim 1,
The host plant is a method of cultivation of a host plant in which plant transformation has been made for manufacturing a plant-based biopharmaceutical, characterized in that the host plant is niventa. 제 2항에 있어서,
상기 제1실내생육공간 또는 제 2실내생육공간의 이산화탄소의 농도는 350 ppm 내지 400ppm 인 것을 특징으로 하는 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법. The method of claim 2,
The method of cultivating a host plant in which plant transformation has been performed for manufacturing plant-based biopharmaceuticals, characterized in that the concentration of carbon dioxide in the first indoor growth space or the second indoor growth space is 350 ppm to 400 ppm. 삭제delete 삭제delete 제 2항 또는 제 3항에 있어서,
상기 기주식물재배단계와 기주식물 회복단계에 있어서, 상기 기주식물을 재배하기 위한 양액의 PH 값을 센서를 이용하여 검출하고, 양액의 PH 값이 5.6 내지 6.9를 유지하도록 하는 PH 조절단계를 더 포함한 것을 특징으로 하는 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법.The method according to claim 2 or 3,
In the host plant cultivation step and the host plant recovery step, the pH value of the nutrient solution for cultivating the host plant is detected using a sensor, and a pH adjustment step of maintaining the pH value of the nutrient solution is 5.6 to 6.9. A method of cultivating a host plant in which plant transformation has been made for manufacturing a plant-based biopharmaceutical, characterized in that 제 1항 또는 제 2항에 있어서,
상기 기주식물재배단계와 기주식물 회복단계에 있어서, 제1,2실내생육공간의 조절된 온도을 온도센서를 이용하여 제1온도검출단계와, 재배판의 배지에 공급되는 양액의 온도를 검출하는 제 2온도검출단계와, 상기 제 1,2온도검출단계에서 검출된 온도를 비교하여 상기 양액의 온도를 제1,2생육공간 내의 온도보다 낮추는 양액온도 조절단계를 더 구비한 것을 특징으로 하는 식물기반 바이오약품 제조를 위한 식물형질전환이 이루어진 기주식물의 재배방법.







The method according to claim 1 or 2,
In the host plant cultivation step and the host plant recovery step, a first temperature detection step using a temperature sensor to control the temperature of the first and second indoor growth spaces, and a temperature of the nutrient solution supplied to the medium of the cultivation plate are detected. Plant-based, characterized in that it further comprises a step of adjusting the temperature of the nutrient solution to lower the temperature of the nutrient solution than the temperature in the first and second growth spaces by comparing the temperature detected in the second temperature detection step and the first and second temperature detection steps. A method of cultivating a host plant that has undergone plant transformation for biopharmaceutical manufacturing.
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