KR20040013424A - Method for mass production of lily - Google Patents
Method for mass production of lily Download PDFInfo
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- KR20040013424A KR20040013424A KR1020020046345A KR20020046345A KR20040013424A KR 20040013424 A KR20040013424 A KR 20040013424A KR 1020020046345 A KR1020020046345 A KR 1020020046345A KR 20020046345 A KR20020046345 A KR 20020046345A KR 20040013424 A KR20040013424 A KR 20040013424A
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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
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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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/60—Flowers; Ornamental plants
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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
- 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
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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
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
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Abstract
Description
본 발명은 백합종구의 대량생산 방법에 관한 것으로, 좀더 상세하게 설명하면 배양단계에 적당한 생물반응기를 적용하여 국외 수입에 주로 의존하는 백합 종구 중 소구에서 중구의 생산을 식물공장식 공정에 의해 규격품을 대량생산 할 수있는 백합종구의 대량생산 방법에 관한 것이다.The present invention relates to a mass production method of lily species, and in more detail, by applying a suitable bioreactor in the cultivation step, the production of junggu in small mouth among the lily species mainly dependent on foreign imports The present invention relates to a mass production method of lily species that can be mass produced.
일반적으로 백합은 바이러스에 감염 될 경우 생장 및 개화 시 꽃의 질에 큰 영향을 미치기 때문에 보편적으로 생장점 배양이라는 조직배양기술을 이용하여 대량증식을 시키고 있다. 조직배양을 실시할 때 대체로 기내에서 생장한 인편이나 기저부를 배양하고 있는데, 이 경우 대량생산이 불가능하고, 배양단계별로 노동집약적인 면이 많이 실제로는 개발도산국 혹은 후진국에서 생산하는 종구에비해 가격 경쟁력이 떨어진다는 것이 주지된 사실이다.In general, lilies are infected with a virus, which greatly affects the quality of flowers during growth and flowering. Therefore, lilies are commonly grown using a tissue culture technique called growth point culture. When tissue culture is carried out, the scales and bases grown on board are generally grown. In this case, mass production is not possible, and the labor-intensive aspects of the culture stages are much higher than those produced in developing or developing countries. It is well known that the competitiveness is low.
특히, 조직배양기술을 이용할 경우 백합 인편배양에서 개화구 생산까지는 약 3년이 소요됨으로써 그 과정에 파생되는 비용을 계산할 경우 농가에서 외국으로부터 직수입하는 것이 더 값싸다는 인식으로 현재 년간 약 50억 이상의 종구가 네델란드로부터 수입되고 있는 실정이다.In particular, using tissue culture technology, it takes about three years from lily cultivation to flowering plant production, and when calculating the costs derived from the process, it is cheaper to import directly from foreign countries. Is imported from the Netherlands.
또한, 수입되는 백합 종구의 가격을 줄이기 위해 일부 농가에서는 수입된 종구의 인편을 야외에서 증식시키는 방법을 사용하고 있으나 이 경우에도 노지에서 생산하는 기간이 약 2년 이상 소요됨으로써 바이러스에 대한 감염율이 높아 생산율이 저하되는 등의 문제가 발생하고 있다.In addition, to reduce the price of imported lily bulbs, some farmers use the method of growing the scales of imported bulbs in the open air, but in this case, the production rate in the open field takes about 2 years or more, so the infection rate is high. Problems such as a decrease in production rate have occurred.
최근에는 생물반응기를 이용하여 백합 종구를 생산하는 방법이 보고된 바 있으나 기저부 배양시 백합의 소구 분리가 어려워 생산성이 급격히 떨어지며, 인편이 포함된 백합소구 역시 기저부 배양과 동일한 문제점을 지니고 있다.Recently, a method of producing a lily species using a bioreactor has been reported, but it is difficult to separate the globules of the lily during the basal culture, and the productivity drops sharply, and the lily globules containing the scales also have the same problems as the basal culture.
특히 기저부 배양이나 인편이 포함된 백합 소구의 경우 20리터 미만의 소형생물반응기 내에서의 배양은 비교적 용이하나 pilot 규모나 산업적인 규모에서는접종에 어려움이 있어 현실적으로 생산단계에 들어간 경우가 더물다.In particular, basal cultures or small globules containing scales are relatively easy to cultivate in small bioreactors of less than 20 liters, but are difficult to inoculate at the pilot scale or industrial scale.
이러한 방법에 있어서 제기되는 또 다른 문제점은 배양 절편 당 궁국적으로 원하는 크기의 백합을 생산할 수 있는 비율이 7개 미만이라는 점에 있다. 이는 다시말해서 생산효율이 감소된다는 것을 의미한다.Another problem posed by this method is that there is less than seven ratios of eventually producing lilies of the desired size per culture section. This means that production efficiency is reduced.
본 발명은 상기와 같은 문제점을 해결하기위해 안출한 것으로써, 백합 기저부 및 인편이 포함된 소구의 배양에 있어서 노동집약적인 생산과정, 종구 생산율 저하, 대량배양에 의한 규격품 생산 공정의 미개발 등을 해결하기 위하여, 인편의 작은 조직을 재료로 하여 이로부터 소구를 생산하는 기술을 개발함으로서 인편 하나 당 소구 생산율을 약 25개로 증식시키기위해 (기존의 5배 이상), 물리적인 조건 중 배양방식과 주입 공기의 효과에 대해 구명하고, 배양 중기에 있어서는 소형 생물반응기에서 대형 생물반응기로 간편하게 이송할 수 있는 시스템 개발과 더불어 배양말기에는 소구의 비대를 위한 최적 조건을 구명함으로써, 최소 경비로 백합 종구 규격품을 대량생산하도록 하는 백합종구의 대량생산방법을 제공하는데 그 목적이 있다.The present invention has been made to solve the above problems, solve the labor-intensive production process, lowering the seed production rate, the non-development of the standard product production process by mass culture in the cultivation of the globules containing the base of the lily and the scales In order to increase the rate of production of globules from about 25 small pieces per piece (more than 5 times), the culture method and inflation air in physical conditions were developed. In the middle of the cultivation, the development of a system that can be easily transferred from the small bioreactor to the large bioreactor, and at the end of the cultivation to find the optimum conditions for the enlargement of the small globules, thereby minimizing the large quantity of lily seed specifications The purpose is to provide a mass production method of lily species to be produced.
상기 목적은 본 발명에 따라, 산소 재순환(Ebb and Flood) 방식의 생물반응기로 소구를 형성하는 단계와, 상기 소구를 산소 및 일반공기가 혼합된 기포발생식 생물반응기를 이용하여 원배치로(one batch) 배양하는 소구유지단계와, 상기 배양물에 일반공기만 주입하여 패드배치(fed batch)로 배양하는 소구비대단계를 포함하는 것을 특징으로 하는 백합종구의 대량생산 방법에 의해 달성된다.According to the present invention, the step of forming a globule with a bioreactor of the oxygen recycle (Ebb and Flood) method, and using the bubble-generating bioreactor mixed with oxygen and the general air to the original batch (one batch) is achieved by a method for mass production of lily seedlings comprising the step of maintaining the globules, and the step of bulging by injecting only normal air into the culture and culturing them in a pad batch.
도 1은 본 발명인 백합종구의 대량생산 방법에 따른 단용 혹은 혼합된 공기 주입을 위해 설계된 기기의 사시도이고,1 is a perspective view of a device designed for single or mixed air injection according to the present invention mass production method of lily species,
도 2는 본 발명인 백합종구의 대량생산 방법에 사용된 공기 재순환식 Ebb and flood 방식의 생물 반응기의 시스템을 나타내는 사시도이며,Figure 2 is a perspective view showing the system of the air recirculation Ebb and flood bioreactor used in the mass production method of the lily species of the present invention,
도 3은 본 발명인 백합종구의 대량생산 방법에 사용된 batch 방식의 기포발생형 생물반응기 시스템을 나타내는 사시도이고,Figure 3 is a perspective view of a batch bubble generation bioreactor system used in the mass production method of the lily species of the present invention,
도 4는 본 발명인 백합종구의 대량생산 방법에 사용된 fed batch 방식의 기포발생형 생물반응기 시스템을 나타낸 사시도이며,Figure 4 is a perspective view showing a bubble batch type bioreactor system fed fed method used in the mass production method of the lily species of the present invention,
도 5는 본 발명인 백합종구의 대량생산 방법에 따라 생산된 백합종구를 나타내는 사진이다.Figure 5 is a photograph showing the lily species produced according to the mass production method of the lily species of the present invention.
이하, 첨부한 도면을 참조하여 본 발명인 백합종구의 대량생산 방법에 관해 좀더 상세하게 설명하면 다음과 같다.Hereinafter, with reference to the accompanying drawings will be described in more detail with respect to mass production method of the present invention lily species.
본 발명은 배양단계별로 상이한 물리적인 환경조건을 적용함으로써 소구에서 중구 크기의 백합 종구 규격품을 대량생산하는 공정 개발에 관한 것이다.The present invention relates to the development of a process for mass production of lily seed standard products of medium size in small mouth by applying different physical environmental conditions for each culture step.
본 발명에서는 초기에 백합 인편 조직으로부터 백합 소구의 대량생산을 위하여 예비실험 결과 가장 효과적인 것으로 나타난 주입 공기에 관한 효과를 검정하기 위해 산소, 이산화 탄소, 질소, 에틸렌 및 일반공기를 각각 단용 혹은 혼용으로 공급하였다. 이때 주입된 공기가 항상 일정한 양으로 유지되도록 하기 위해 재순환 방식의 생물반응기를 설계, 제작하여 사용하였다. 백합 인편 조작을 배양하여 대량의 소구가 생성된다고 하더라도, 생산괸 소구가 서로 뭉쳐서 생장함으로 인해 소형생물반응기에서 대형생물반응기로의 이송이 불가능하다. 이러한 점을 개선하기 위해, 소형 생물반응기에 이송용 포트를 설치하고 배양 방법적으로는 2일 간격으로 배양액을 주입하여 배양체를 분리시키는 공정을 채택하였다. 소구의 비대의 생산과 비대는 완전히 다른 공정임으로 배양말기에는 소구의 비대에 fed batch 방식의 기포발생형 생물반응기를 이용하였으며, 화학적인 배지 조성을 달리하여 규격품의 백합 종구 생산이 가능하도록 하였다.In the present invention, initially, oxygen, carbon dioxide, nitrogen, ethylene and general air are supplied singly or in combination to test the effect on the injected air which is shown to be most effective as a result of preliminary experiments for mass production of lily globules from lily scaly tissue. It was. At this time, the recycled bioreactor was designed, manufactured and used to maintain the injected air at a constant amount. Even if a large number of globules are produced by cultivating lily scales, it is impossible to transfer them from a small bioreactor to a large bioreactor due to agglomeration and growth of production globules. In order to improve this point, a small bioreactor was equipped with a transfer port, and the culture method was adopted to separate the culture by injecting the culture solution every two days. The production and enlargement of the globules is a completely different process. At the end of the incubation, a bubbled bioreactor of fed batch type was used for the globules of the globules.
백합 종구의 생산효율 면에서 볼 때, 기존의 조직배양법을 이용할 경우 100개의 시료로 시작하여 1차 계대배양 시 약 7배의 증식율로 계산하면, 5차 계대배양 시 이론적으로 약 1,680,700 개의 백합 종구 생산이 가능 한 반면, 본 발명에 의해생산할 경우에는 동일한 조건하에서 5차 계대배양 시 약 976,562,500 개의 백합 종구 생산이 가능함으로써 약 581배의 높은 효율을 기대할 수 있다.In terms of the production efficiency of lily seedlings, using the conventional tissue culture method, starting with 100 samples and calculating the growth rate of about 7 times at the first passage, the theoretical production of 1,680,700 lily seedlings at the 5th passage is theoretically produced. On the other hand, in the case of production according to the present invention, it is possible to produce about 976,562,500 lily seed bulbs during the fifth subculture under the same conditions, so that a high efficiency of about 581 times can be expected.
이러한 수치는 계대배양 시 시료를 완전히접종할 경우에 가능한데 기존의 조직배양 시 1,680,700 개의 백합 소구를 생산하기위해 약 20명 이상의 상주 인력이 필요한 반면, 본 발명에 나타난 방법으로 실시할 경우 5명 내외의 인원으로 충분하다. 백합 소구를 비대하여 중구 크기의 종구를 대량생산하는 측면에서 볼 때 일반 조직배양방법은 현실적으로 불가능에 가깝다고 할 수 있으나, 본 발명에 의할 경우 대형 생물반응기를 설치하여 한번의 접종으로 가능하다. 조직배양에의해 생산된 종구를 비대하기 위해서는 일반적으로 야외에서 소구를 재배하는 방법을 이용하고 있는데 이 경우 기간이 약 2년 소요되며 바이러스의 감염율이 높을 수 가있으나, 본 발명에서 처럼 단계별로 적절한 생물반응기를 사용할 경우 그 기간을 6개월로 단축할 뿐만아니라 소구 비대과정 중에 바이러스 감염율은 거의 제로에 가까운 수준으로 유지시킬 수 있다.These values are available for complete inoculation of the sample at subculture, whereas more than 20 resident personnel are required to produce 1,680,700 lily globules for conventional tissue culture, while the method shown in the present invention is about 5 persons. The staff is enough. In view of the mass production of neutrophils in the size of lily globules, the general tissue culture method can be said to be practically impossible, but in accordance with the present invention, it is possible to install a large bioreactor with a single inoculation. In order to enlarge the species produced by tissue culture, generally, a method of cultivating outdoor globules is used. In this case, it takes about 2 years and the infection rate of the virus may be high. The use of the reactor not only shortens the period to six months, but also keeps the virus infection rate near zero during the enlargement process.
[시험예1][Test Example 1]
배양단계별로 상이한 물리적인환경조건을 적용하여 백합 종구를 대량으로 생산하는 방법은 다음과 같다. 백합 개화구의 생장점을 배양하여 이로부터 바이러스 무병주 식물을 기내에 확립한다. 이때 배양배지는 일반적으로 사용되고 있는 식물조직배양용 배지를 사용하였으며, 배지는 산도를 5.6으로 조절하고, 한천을 0.75%, 설탕을 약 3%, 그리고 식물생장조절물질 중 오옥신을 소량 첨가하여 굳힌 고체배지를 이용하였다. 배양기는 녹십자에서 공급되는 일회용 플라스틱 접식 (직경 9cm)를 사용하였으며, 배양환경은 25℃로 조절된 암상태에서 24시간 유지시켰다. 인편이 약 5에서 7장 달린 소구가 형성되었을 , 인편을 한 장씩 분리하여 전술한 동일배지에 사이토키닌이 소량 넣어 배양하였을 때, 3에서 10개의 소구가 생산되었다. 생산된 소구를 하나씩 분리하여 식물생장조절물질이 거의 함유되지 않은 배지에 2에서 3개월 배양할 경우 다시 4에서 7장의 인편이 달린 구로 생장을 하였다. 인편으로부터 소구유도와 소구부대 과정을 약 6에서 7회 반복 실시하여 충분한 개수의 시료를 준비하였다. 초기단계에서 주입공기에 따라 소구의 생산성을 계산하기 위하여 Ebb and flood 방식의 소형 생물반응기에 공기 제순환을 할 수 있는 보조탱크를 설치한 후 이들을 121℃로 멸균하여 배양을 위해 준비하였다. 배양을 위한 시료를 조제하기 위해 조직배양된 인편을 칼로 잘게 설여서 1개의 인편당 약 30개의 조직이 나오도록 준비하였다. 이때 한천을 함유하지 않은 동일배지를 액체 상태로 조제하여 멸균하여 준비하였다. 준비된 배양체와 배양배지를 생물반응기에 접종한 다음 감압기로 생물반응기와 보조탱크의 공기를 일정량 제거시킨 후 다시 시험에 사용된 공기를 주입시킴으로 인해, 보조탱크에 일정량이 공기가 주입되면 주입된 공기 체적만킁의 배양재지가 생물반응기속으로 이동되며, 생물반응기내에 존재하던 공기 중 배양배지 만큼이 체적이 생물반응기로부터 보조탱크로 주입되고, 타이마에 의해 생물반응기내의 배지가 보조탱크로 다시 내려갈 때, 생물반응기로부터 보조탱크로 이동하는 배지 체적 만큼의 공기가 다시 보조탱크에서 생물반응기로 삽입하도록 함으로써 완전한 재순환이 가능하도록 하였다. 배양 약 2주 후부터 배양된 인편조각으로부터 소구가 형성되기 시작하는데, 그대로 방치해 둘 경우 수구에서 유도된 뿌리가 엉키여 소형생물반응기에서 다음단계인 중형 생물반응기로의 이송을 불가능하게 만들므로 이를 해결하기 위해 3일에 한번씩 보조탱크 속의 배양배지를 인위적으로 소형 생물반응기내로 모두 주입시킨 후 공기량이 약 1.0 vvm이되도록 함으로써 배양상태의 소구가 풀어지도록 하였다. 소구가 형성된 후에는 특정공기가 불필요함으로써 외부의 일반공기를 사용하는 방식의 생물반응기를 제작하였으며, 이때 생물반응기는 기포발생형으로 설계하였으며 배지공급은 batch 방식으로 실시하였다. 소구의 비대를위해 배양말기에는 배양중기에서 사용한 것과 동일한 기포발생형 생물반응기를 사용하였으나, 이때에는 fed batch 방식으로 배지를 공급하는 방법을 채택하여 소구의 비대를 도모하였다.The method of producing large quantities of lily seeds by applying different physical environmental conditions for each culture step is as follows. The growth point of lily blooms is incubated to establish virus-free plants on board. At this time, the culture medium was used for plant tissue culture medium which is generally used, the medium was adjusted to acidity of 5.6, 0.75% agar, about 3% sugar, and solidified by adding small amounts of oxine in plant growth regulators. Medium was used. The incubator used a disposable plastic grafting (diameter 9 cm) supplied from the green cross, the culture environment was maintained for 24 hours in the dark condition adjusted to 25 ℃. When five to seven globules of scales were formed, three to ten globules were produced when the scales were separated one by one and cultured with a small amount of cytokinin in the same medium described above. When the produced globules were separated one by one and cultured for two to three months in a medium containing little plant growth regulators, they were grown to four to seven pieces of spheres. A sufficient number of samples were prepared by repeating the globule induction and globule bag process from about 6 to 7 times. In order to calculate the productivity of the globules according to the inlet air in the initial stage, after installing the auxiliary tank for air recirculation in a small bioreactor of Ebb and flood method, they were sterilized at 121 ° C and prepared for incubation. In order to prepare a sample for cultivation, the tissue cultured scales were cut finely with a knife to prepare about 30 tissues per piece. At this time, the same medium containing no agar was prepared by sterilization in a liquid state. After inoculating the prepared culture and culture medium into the bioreactor, and after depressurizing the air of the bioreactor and the auxiliary tank with a depressurizer, the air used for the test is injected again. Ten thousand cultures are transferred into the bioreactor, and as much of the culture medium in the bioreactor as the volume is injected from the bioreactor into the auxiliary tank, and by Tyma, the medium in the bioreactor is lowered back into the auxiliary tank. As much as the volume of the medium moving from the bioreactor to the auxiliary tank was inserted back into the bioreactor from the auxiliary tank to allow for complete recirculation. After about two weeks of culture, globules begin to form from the cultured scaffolds. If left untreated, the roots derived from the polo tang are entangled, making it impossible to transfer them from the small bioreactor to the next stage of the medium bioreactor. To this end, every three days, the culture medium in the auxiliary tank was artificially injected into the small bioreactor and the air volume was about 1.0 vvm to release the cultured globules. After the formation of the globules, there was no need for specific air so that a bioreactor was constructed using external general air. At this time, the bioreactor was designed to be bubble-producing, and the medium was supplied in a batch method. For the enlargement of the globules, the same bubble-generating bioreactor was used at the end of the culture period, but at this time, the globules were enlarged by adopting a fed batch method.
본 발명인 백합종구 대량생산방법은 생산단계별로 적절한 생물반응기를 사용하기때문에 소구재배기간이 단축되고, 소구비대과정 중 바이러스 감염이 없어 생산율이 증가하는 등, 최소경비로 백합 종구의 규격품을 대량생산할 수 있어 수입에 의존하던 백합종구의 생산 및 해외수출이 가능하기 때문에 백합종구를 생산하는 농가의 소득을 증가시킬 수 있을 뿐만아니라 소비자의 효율성을 극대화 하는 매우 훌륭한 발명이다.In the present invention, the mass production method of lily seed ball can be mass-produced standard products of lily seed ball with minimum cost because the use of appropriate bioreactor for each production stage shortens the globule cultivation period and increases the production rate because there is no virus infection during the globule enlargement process. As it is possible to produce and export overseas lily species, which is dependent on imports, it is an excellent invention that can not only increase the income of farmers who produce lily species but also maximize the efficiency of consumers.
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Cited By (5)
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| CN103283604A (en) * | 2013-06-07 | 2013-09-11 | 南京工业大学大丰海洋产业研究院 | Method for removing endophytic bacteria of explants in lily tissue culture |
| CN103636502A (en) * | 2013-12-11 | 2014-03-19 | 广西壮族自治区农业科学院生物技术研究所 | Method for accelerating expansion growth of bulbs during Lanzhou lily tissue culture |
| CN103688864A (en) * | 2013-12-18 | 2014-04-02 | 杭州市园林绿化股份有限公司 | Lilium oriental hybrid tissue culture seedling strengthening method |
| CN104067937A (en) * | 2014-06-23 | 2014-10-01 | 云南省农业科学院花卉研究所 | Tide type bioreactor and method for culturing lily bulblets thereof |
| KR102243587B1 (en) * | 2020-07-27 | 2021-04-22 | 주식회사 네이처농업회사법인 | Apparatus for producing lilies in large quantities |
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| CN103636502B (en) * | 2013-12-11 | 2015-10-28 | 广西壮族自治区农业科学院生物技术研究所 | A kind of promote lanzhou lily group train bulb expand grow method |
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| KR102243587B1 (en) * | 2020-07-27 | 2021-04-22 | 주식회사 네이처농업회사법인 | Apparatus for producing lilies in large quantities |
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