KR100497717B1 - Method of culturing somatic embryo of broccoli and method for the mass-production of broccoli sprouts using the same - Google Patents
Method of culturing somatic embryo of broccoli and method for the mass-production of broccoli sprouts using the same Download PDFInfo
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- KR100497717B1 KR100497717B1 KR10-2002-0066165A KR20020066165A KR100497717B1 KR 100497717 B1 KR100497717 B1 KR 100497717B1 KR 20020066165 A KR20020066165 A KR 20020066165A KR 100497717 B1 KR100497717 B1 KR 100497717B1
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- broccoli
- plant
- culturing
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Classifications
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- 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
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
본 발명은 브로콜리의 체세포배 배양 방법 및 이를 이용한 브로콜리 식물체싹의 대량 생산 방법에 관한 것으로서, 보다 상세하게는 1) 배를 형성할 수 있는 브로콜리의 조직을 배 유기용 배지에 배양하여 배를 유기시키는 단계; 2) 단계 1의 배를 절단하여 캘러스 형성 배지에 치상한 후 배양하여 캘러스를 형성시키는 단계; 3) 단계 2의 캘러스로부터 배발생 세포를 형성시키는 단계; 및 4) 단계 3의 배발생 세포를 호르몬이 제거된 배지에 옮겨 계대배양을 실시하는 단계를 포함하는 브로콜리의 체세포배 배양 방법 및 이를 이용한 브로콜리 식물체싹의 대량 생산 방법에 관한 것이다. 본 발명의 브로콜리의 체세포배 배양 방법은 종자로부터 얻을 수밖에 없었던 브로콜리의 식물체싹을 체세포를 이용하여 대량 생산하게 함으로써 짧은 시간 내에 간편하게 무한정으로 브로콜리의 식물체싹을 생산할 수 있으며, 상기의 방법으로 생산된 브로콜리의 유식물체는 항암제성분인 설포라판이 다량 함유되어 있으므로 항암용 건강식품 또는 의약품의 원료로써 값싸게 사용될 수 있을 뿐만 아니라, 브로콜리의 형질전환 등과 같은 생명공학 기술에 매우 유용하게 사용될 수 있다.The present invention relates to a method for culturing somatic embryos of broccoli and a method for mass production of broccoli plant buds using the same, and more specifically, 1) organizing embryos by culturing a tissue of broccoli capable of forming pears in a pear organic medium. step; 2) cutting the pear of step 1, soaking in callus forming medium, and incubating to form callus; 3) forming embryogenic cells from the callus of step 2; And 4) relates to a method of culturing broccoli somatic embryos comprising the step of transferring the embryogenic cells of step 3 to a medium from which the hormone is removed, and a mass production method of broccoli plant shoots using the same. The method of culturing the somatic embryos of broccoli of the present invention allows the mass production of plant seeds of broccoli, which had to be obtained from seeds, by mass production using somatic cells, so that plant sprouts of broccoli can be easily and infinitely produced in a short time, and broccoli produced by the above method. Since the seedlings of the plant contain a large amount of anti-cancer drug sulforaphane, it can be used inexpensively as a raw material of anti-cancer health foods or medicines, and can be very useful for biotechnology such as transformation of broccoli.
Description
본 발명은 브로콜리의 체세포배 배양 방법 및 이를 이용한 브로콜리 식물체싹의 대량 생산 방법에 관한 것으로서, 보다 상세하게는 종자로부터 얻을 수밖에 없었던 브로콜리의 식물체싹을 체세포를 이용하여 대량 생산하게 함으로써 짧은 시간 내에 간편하게 무한정으로 브로콜리의 식물체싹을 생산할 수 있는 브로콜리의 체세포배 배양 방법 및 이를 이용한 브로콜리 식물체싹의 대량 생산 방법에 관한 것이다.The present invention relates to a method for cultivating somatic embryos of broccoli and a method for mass production of broccoli plant buds using the same, and more specifically, to allow broccoli plant sprouts, which had to be obtained from seeds, to be mass-produced using somatic cells in a simple and indefinite time. It relates to a somatic embryo culture method of broccoli that can produce plant buds of broccoli and a mass production method of broccoli plant buds using the same.
암은 미국에서 두 번째로 사망률이 높은 질환이며, 해마다 사망하는 사람들 중 거의 1/4이 암으로 사망한다고 알려져 있다(Parker S.L., et al., Ca Cancer J. clin., 1997, 47, 5-27). 어떤 종류의 암의 경우에는 발생율이 줄어들고 있기도 하지만, 다른 암의 경우에는 발생율이 증가한다. 따라서, 초기에 진단하고 치료하는 기술이 발전하였음에도 불구하고, 전체적인 암 사망률은 1970년대 이후로 거의 변화되지 않고 있다. 따라서, 암의 예방에 관한 더 많은 연구가 요구되고 있는 실정이다(Bailar J.C., N. Engl. J. Med., 1997, 336, 1569-1574). 지난 20년 동안 암을 예방하기 위한 식생활로서 과일과 야채의 섭취가 강조되어 왔으며(Nestle M., J. Natl. Cancer Inst. Monogr., 1992, 12, 153-157), 최근에는 표 1에 나열된 것과 같은 식물에 기초한 섭취에 높은 우선권을 부여하고 있다(American Cancer Society Advisory Committee on Diet, Nutrition, and Cancer Prevention, Ca Cancer J. Clin., 1996, 46, 325-341; World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer, 1997).Cancer is the second highest mortality disease in the United States, with nearly one in four deaths reported annually (Parker SL, et al., Ca Cancer J. clin. , 1997 , 47, 5- 27). The incidence rate is decreasing in some types of cancer, while incidence increases in other cancers. Thus, despite advances in early diagnosis and treatment, overall cancer mortality has changed little since the 1970s. Therefore, more research on the prevention of cancer is required (Bailar JC, N. Engl. J. Med. , 1997 , 336, 1569-1574). Fruit and vegetable intake has been emphasized in the past 20 years as a diet to prevent cancer (Nestle M., J. Natl. Cancer Inst. Monogr. , 1992 , 12, 153-157), recently listed in Table 1 High priority for plant-based intake, such as the American Cancer Society Advisory Committee on Diet, Nutrition, and Cancer Prevention, Ca Cancer J. Clin. , 1996 , 46, 325-341; World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer , 1997 ).
상기와 같은 지침들은 심장질환과 같은 식이와 관련되는 다른 만성질환을 예방하기 위한 권고안과 전적으로 일치하며(Nestle M., J. Natl. Cancer Inst. Monogr., 1992, 12, 153-157), 이것은 식물의 섭취와 몇몇 주요 부위에서의 암 발생율 감소의 연관성을 보여주는 강력한 증거들에 의해서 확인된다(World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer, 1997; Block G., et al., Nutr. Cancer, 1992, 18, 1-29; Steinmetz K.A., et al., Cancer Causes Control, 1991, 2, 325-357). 상기와 같은 사실에 근거하여, 식물에 기초한 식이를 함으로써 20% (World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer, 1997) 내지 50% (Steinmetz K.A., 1996, J. Am. Diet. Assoc., 1996, 96, 1027-1039) 정도의 암 발생을 억제할 수 있을 것으로 추정되고 있다.These guidelines are in full agreement with recommendations for preventing other chronic diseases associated with diet such as heart disease (Nestle M., J. Natl. Cancer Inst. Monogr. , 1992 , 12, 153-157). This is confirmed by strong evidence showing the association between plant intake and reduced cancer incidence in several key areas (World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer , 1997 ; Block G., et al., Nutr. Cancer , 1992 , 18, 1-29; Steinmetz KA, et al., Cancer Causes Control , 1991 , 2, 325-357). Based on the above facts, 20% (World Cancer Research Fund and American Institute for Cancer Research, Food, Nutrition and the Prevention of Cancer , 1997 ) to 50% (Steinmetz KA, 1996, J. Am. Diet.Assoc . , 1996 , 96, 1027-1039).
역학조사 및 동물 모델 연구로부터, 몇몇 식물들이 암의 발생 위험을 현저하게 감소시킨다는 것이 알려져 있다. 이러한 식물들 중에는 배추과(cruciferous) 식물에 속하는 브로콜리, 양배추, 꽃양배추(cauliflower) 및 브루쎌 식물체싹(Brussels sprout)과 같은 브라시카 속(Brassica genus)의 식물들이 있다. 미국 국가 위원회에서는 1980년대부터 암의 발생을 방지하기 위하여 이러한 식물들을 섭취하도록 권고해왔다(National Research Council, Diet, Nutrition, and Cancer, 1982).From epidemiological studies and animal model studies, it is known that some plants significantly reduce the risk of developing cancer. Among these plants are plants of the Brassica genus, such as broccoli, cabbage, cauliflower and Brussels sprout, belonging to the cruciferous plant. The US National Council has recommended eating these plants since the 1980s to prevent the development of cancer (National Research Council, Diet, Nutrition, and Cancer , 1982 ).
브라시카 속의 식물들은 낮은 지방 함유량을 보이며, 에너지가 낮고, 비타민, 미네랄 및 식이섬유가 다량 함유되어 있다. 이러한 모든 특성들은 암 발생의 억제와 관련이 있다. 또한, 이들은 많은 양의 식물화학물질을 포함하는데, 이들 중 어떤 것들은 다양한 시험관내(in vitro) 및 동물 모델에서 암의 발생을 억제한다고 알려져 있다(Steinmetz K.A., 1996, J. Am. Diet. Assoc., 1996, 96, 1027-1039).Plants in the genus Brassica are low in fat, low in energy and high in vitamins, minerals and fiber. All these properties are related to the inhibition of cancer development. They also contain large amounts of phytochemicals, some of which are known to inhibit the development of cancer in a variety of in vitro and animal models (Steinmetz KA, 1996, J. Am. Diet.Assoc. , 1996 , 96, 1027-1039).
파헤이 등의 연구는 브라시카 속의 식물에 존재하는 특정한 식물화학물질을 동정하는데 맞춰져 있는데(Fahey J.W., et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 10367-10372), 상기 연구에서는 식물화학물질이 효소 시스템의 활성을 유도하여 발암물질을 중화시킨다고 보고하고 있다. 몇몇 효소 시스템은 산화, 환원 또는 가수분해에 관여하며(phase 1), 이후 약재, 대사산물, 발암물질 및 다른 독성 화합물과 결합하거나 또는 다른 방법으로 영향을 미쳐(phase 2) 이들의 극성(polarity) 및 배출성(excretability)을 증가시킨다. 페이스 1 효소들은 실험 조건에 따라서 발암물질들을 활성화시키거나 또는 불활성화시키며, 페이스 2 효소들은 독성을 더 많이 중화시킨다. 현재까지, 배추과의 식물들을 섭취하면 실험적인 시스템에서 효소의 해독(detoxification)을 유도하는 것으로 알려져 있다(National Research Council, Diet, Nutrition, and Cancer, 1982).Fahey et al.'S work is focused on identifying specific phytochemicals present in plants in the genus Brassica (Fahey JW, et al., Proc. Natl. Acad. Sci. USA , 1997 , 94, 10367-10372). Reports that phytochemicals neutralize carcinogens by inducing the activity of enzyme systems. Some enzymatic systems are involved in oxidation, reduction or hydrolysis (phase 1), and then combine or otherwise affect the medicinal agents, metabolites, carcinogens and other toxic compounds (phase 2) And increase excretability. Phase 1 enzymes activate or deactivate carcinogens, depending on experimental conditions, and phase 2 enzymes neutralize more toxicity. To date, ingestion of cabbage plants is known to induce detoxification of enzymes in experimental systems (National Research Council, Diet, Nutrition, and Cancer , 1982 ).
현재, 페이스 1 및 페이스 2 효소 시스템 모두를 유도할 수 있는 양기능성(bifunctional) 식물화학물질을 페이스 2 효소만을 유도하는 단일기능성(monofunctional) 식물화학물질로부터 구별하기 위한 시험관내 분석 방법을 개발하였으며, 상기 분석방법을 이용하여 브라시카 식물체가 단일기능성 페이스 2 유도자(inducer)를 특별히 풍부하게 함유하고 있고(Prochaska H.J., Proc. Natl. Acad. Sci. USA, 1992, 89, 2394-2398), 이소티오시아네이트 설포라판(isothiocyanate sulforaphane)이 브로콜리 추출물에서의 주된 페이스 2 유도자임을 밝혔다(Zhang Y., et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 2399-2403). 또한, 이들은 설포라판이 래트에서 발암물질에 의해서 유도되는 종양의 발생을 농도-의존적으로 억제한다는 것을 확인하였다(Zhang Y., et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 3147-3150).Currently, we have developed an in vitro assay to distinguish bifunctional plant chemicals capable of inducing both phase 1 and face 2 enzyme systems from monofunctional plant chemicals that only induce phase 2 enzymes. Using this assay method, the Brassica plant contains abundantly monofunctional Phase 2 inducers (Prochaska HJ, Proc. Natl. Acad. Sci. USA , 1992 , 89, 2394-2398), isothio Isothiocyanate sulforaphane was found to be the major Phase 2 inducer in broccoli extracts (Zhang Y., et al., Proc. Natl. Acad. Sci. USA , 1992 , 89, 2399-2403). In addition, they confirmed that sulforaphane concentration-dependently inhibited the development of tumor-induced tumors in rats (Zhang Y., et al., Proc. Natl. Acad. Sci. USA , 1994 , 91, 3147). -3150).
이러한 활성의 대부분은 설포라판 및 글루코라파닌(glucoraphanin)의 글루코시놀레이트(glucosinolate) 전구체로부터 유래된 것이다. 식물체싹 이후에는 페이스 2 효소의 합성이 일어나지 않기 때문에, 식물이 성장할수록 이들 전구체의 농도는 감소된다. 브로콜리 식물체싹의 추출물은 성숙한 브로콜리 식물체보다 페이스 2 유도자 활성이 10-100배 더 높으며, 래트에서의 종양발생을 더 효과적으로 억제하였다. 반면에, 성숙한 브로콜리는 페이스 2 뿐만 아니라 페이스 1 효소도 유도하는 인돌 화합물을 상당량 함유하고 있다. 따라서, 식물체싹은 항암활성에 있어서 성숙한 브로콜리보다 적어도 두가지 장점을 더 가지고 있는 것으로 보인다. 즉, 브로콜리 식물체싹은 높은 농도의 유도자를 포함하고 있으며, 또한 상기 유도자는 페이스 2 효소 시스템에 주로 작용한다. 상기 사실에 기초하여, 파헤이 등은 작은 양의 배추과 식물체의 식물체싹은 동일한 종류의 많은 양의 성숙한 식물체의 경우와 동일한 항암 활성을 가지고 있다고 결론지었다.Most of this activity is derived from the glucosinolate precursors of sulforaphane and glucoraphanin. Since plant synthesis does not occur after plant shoots, the concentration of these precursors decreases as the plant grows. Extracts of broccoli plant buds were 10-100 times higher in phase 2 inducer activity than mature broccoli plants and more effectively inhibited tumorigenesis in rats. In contrast, mature broccoli contains significant amounts of indole compounds that induce not only face 2 but also face 1 enzymes. Thus, plant buds appear to have at least two more advantages than mature broccoli in anticancer activity. That is, broccoli plant shoots contain high concentrations of inducers, which also act primarily on the Phase 2 enzyme system. Based on the above facts, Fahey et al. Concluded that the plant buds of small cabbages and plants have the same anticancer activity as in the case of large amounts of mature plants of the same kind.
브로콜리는 배추과 작물의 하나로서 채소로 이용될 뿐만 아니라 브로콜리 내에 함유하고 있는 설포라판(Sulforaphane)은 항암물질(Proc. Natl. Acad. Sci. USA, 1997, 94, 10367-10372)과 위내에서 위암을 일으키는 헬리코박터 세균을 특이적으로 죽인다는 연구 결과가 보고되어 그 효과가 탁월하다는 것이 입증되었다(Proc. Natl. Acad. Sci. USA, 2002, 99, 7610-7615). 브로콜리 내에 있는 설포라판이라는 물질은 브로콜리 뿐만 아니라 배추과 작물인 배추, 무 등에서도 함유되어 있으나 그 함유량이 극히 미미한 편이며 브로콜리 내에서도 식물체싹이 다른 부위보다 50-100배정도 더 높은 설포라판을 함유하고 있다(Marion Nestle, Proc. Natl. Acad. Sci. USA, 1997, 94, 11149-11151). 설포라판은 화학적으로 합성하기가 어렵고, 식물체싹은 종자로부터 생산되기 때문에 그 가격이 매우 높아서, 많은 암환자나 암예방을 원하는 사람들에게 충분히 보급되지 못하는 실정이다.Broccoli is not only used as a vegetable as a cabbage crop, but also Sulforaphane contained in broccoli is an anticancer substance ( Proc. Natl. Acad. Sci. USA , 1997 , 94, 10367-10372) and gastric cancer in the stomach. Studies on the specific killing of Helicobacter bacteria have been reported, demonstrating their effectiveness ( Proc. Natl. Acad. Sci. USA , 2002 , 99, 7610-7615). The substance called sulforaphane in broccoli is found not only in broccoli, but also in Chinese cabbage and Chinese cabbage, radish, etc., but the content is very small, and in broccoli, plant buds contain about 50-100 times higher than other parts (Marion Nestle) Proc. Natl. Acad. Sci. USA , 1997 , 94, 11149-11151). Sulforapane is difficult to synthesize chemically, and since plant shoots are produced from seeds, the price is very high, so that many cancer patients or people who want cancer prevention are not widely distributed.
한편, 종자로부터 배를 발생시키는 전통적인 방법 외에 체세포를 이용한 배발생법에 의한 유식물체 생산은 가시오갈피, 인삼 등에서 이미 보고되었으나, 브로콜리의 경우에는 아직 보고된 바가 없다(Plant Cell Reports, 1991, 9, 514-516; Korean J. Plant Tissue Culture, 1993, 20, 216-266).On the other hand, in addition to the traditional method of generating embryos from seeds, seedling production by embryogenic methods using somatic cells has already been reported in psoriasis and ginseng, but broccoli has not been reported yet ( Plant Cell Reports , 1991 , 9, 514-516; Korean J. Plant Tissue Culture , 1993 , 20, 216-266).
이에, 본 발명자들은 브로콜리의 약(anther)을 이용한 브로콜리의 체세포배 배양 방법을 개발하였고, 상기 방법을 이용하여 설포라판의 함유량이 뛰어난 브로콜리의 식물체싹을 대량으로 생산함으로써 지금까지 종자로부터만 생산할 수 있어서 고가였던 브로콜리의 식물체싹을 매우 경제적으로 저렴하게 생산할 수 있으며, 상기 브로콜리 식물체싹을 직접 또는 가공하여 건강식품 또는 의약품으로 사용할 수 있음을 밝힘으로써 본 발명을 완성하였다.Therefore, the present inventors have developed a method of culturing broccoli somatic embryos using broccoli's anther, and by using the above method, the plant buds of broccoli having excellent sulforaphane content can be produced in large quantities so far only from seeds. The present invention was completed by revealing that the plant bud of broccoli, which was expensive, can be produced very economically and inexpensively, and that the broccoli plant sprout can be directly or processed to be used as a health food or medicine.
본 발명은 브로콜리의 체세포배 배양 방법 및 이를 이용한 브로콜리 식물체싹의 대량 생산 방법을 제공하는 것이다. The present invention provides a method for culturing somatic embryos of broccoli and a mass production method of broccoli plant shoots using the same.
본 발명의 다른 목적은 브로콜리의 식물체싹을 포함하는 암의 예방 또는 치료용 건강식품 및 약학적 조성물을 제공하는 것이다. Another object of the present invention to provide a health food and pharmaceutical composition for the prevention or treatment of cancer, including plant shoots of broccoli.
상기 목적을 달성하기 위하여, 본 발명은 1) 배를 형성할 수 있는 브로콜리의 조직을 배 유기용 배지에 배양하여 배를 유기시키는 단계; 2) 단계 1의 배를 절단하여 캘러스 형성 배지에 치상한 후 배양하여 캘러스를 형성시키는 단계; 3) 단계 2의 캘러스로부터 배발생 세포를 형성시키는 단계; 및 4) 단계 3의 배발생 세포를 호르몬이 제거된 배지에 옮겨 계대배양을 실시하는 단계를 포함하는 브로콜리의 체세포배 배양 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of: 1) cultivating the tissue of broccoli that can form a pear in pear organic medium to organic pear; 2) cutting the pear of step 1, soaking in callus forming medium, and incubating to form callus; 3) forming embryogenic cells from the callus of step 2; And 4) transferring the embryogenic cells of step 3 to a medium from which the hormone has been removed, thereby performing subculture.
또한, 본 발명은 상기 체세포배 배양 방법을 이용한 브로콜리 식물체싹의 대량 생산방법을 제공한다.The present invention also provides a mass production method of broccoli plant shoots using the somatic embryo culture method.
또한, 본 발명은 상기의 방법으로 제조된 브로콜리의 식물체싹을 포함하는 암의 예방 또는 치료용 건강식품을 제공한다.In another aspect, the present invention provides a health food for the prevention or treatment of cancer comprising a plant shoot of broccoli prepared by the above method.
또한, 본 발명은 상기의 방법으로 제조된 브로콜리의 식물체싹을 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, including plant shoots of broccoli prepared by the above method.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 브로콜리의 체세포배 배양 방법을 제공한다.The present invention provides a method for culturing somatic embryos of broccoli.
본 발명의 브로콜리의 체세포배 배양 방법은Somatic embryo culture method of broccoli of the present invention
1) 배를 형성할 수 있는 브로콜리의 조직을 배 유기용 배지에 배양하여 배를 유기시키는 단계;1) culturing a tissue of broccoli capable of forming a pear in a pear organic medium to organic a pear;
2) 단계 1의 배를 절단하여 2,4-D를 포함하는 캘러스 형성 배지에 치상한 후 배양하여 캘러스를 형성시키는 단계;2) cutting the embryo of step 1 and incubating in a callus forming medium including 2,4-D, followed by culturing to form callus;
3) 단계 2의 캘러스로부터 배발생 세포를 형성시키는 단계; 및3) forming embryogenic cells from the callus of step 2; And
4) 단계 3의 배발생 세포를 호르몬이 제거된 배지에 옮겨 계대배양을 실시하는 단계를 포함한다.4) transferring the embryogenic cells of step 3 to a medium from which the hormone has been removed, and performing subculture.
단계 1에 있어서, 배를 형성할 수 있는 브로콜리의 조직은 브로콜리의 종자, 약(anther), 뿌리, 줄기 및 잎으로 구성된 군으로부터 선택될 수 있으며, 이 중에서 브로콜리의 종자 또는 약인 것이 바람직하고, 브로콜리의 약인 것이 더욱 바람직하다. 본 발명에서는 바람직한 실시예로서 약을 사용하여 배양한 경우를 예시하였다. 브로콜리의 약은 모든 발생단계의 약을 사용할 수 있으나, 1핵기 전후 시기의 소포자를 가지는 약을 사용하는 것이 더욱 바람직하다. 배 발생용 배지는 MS 배지(Duchefa M0231), Gamborg B5 배지(Duchefa G0210) 및 하기 표 2의 조성을 가지는 B5 수정배지로 구성된 군으로부터 선택될 수 있으며, 이 중에서 B5 수정배지를 사용하는 것이 바람직하다.In step 1, the tissue of broccoli capable of forming a pear may be selected from the group consisting of seeds, anthers, roots, stems and leaves of broccoli, preferably the seeds or medicaments of broccoli, preferably broccoli It is more preferable to be a medicine. In the present invention, the case of culturing using a medicine as a preferred embodiment is illustrated. The drug of broccoli may be used at all developmental stages, but it is more preferable to use a drug having a vesicle at a time before and after the nuclear stage. Embryo development medium may be selected from the group consisting of MS medium (Duchefa M0231), Gamborg B5 medium (Duchefa G0210) and B5 fertilization medium having the composition of Table 2 below, it is preferable to use a B5 fertilization medium.
B5 수정배지를 사용하는 경우 0.1 ㎎/ℓ NAA, 0.1 ㎎/ℓ 2,4-D 및 125 ㎎/ℓ AgNO3를 포함하는 것이 가장 바람직하지만 상기 농도는 조건에 따라서 어느 정도 변화될 수 있음은 당업자에게 있어서는 자명하다.It is most preferable to include 0.1 mg / l NAA, 0.1 mg / l 2,4-D and 125 mg / l AgNO 3 when using a B5 fertilization medium, but the concentration may vary to some extent depending on the conditions. It is self-evident to.
단계 2에 있어서, 캘러스 형성 배지는 Chu(N6)(Duchefa C0204), Gamborg B5 및 MS 배지로 구성된 군으로부터 선택될 수 있으며, 이 중에서 MS 배지를 사용하는 것이 바람직하다. MS 배지를 사용하는 경우 2,4-D가 포함되는 것이 바람직하며, 이때 0.1 내지 1.0 ㎎/ℓ의 농도가 더욱 바람직하고 0.2 내지 0.5 ㎎/ℓ의 농도인 것이 가장 바람직하다.In step 2, the callus forming medium may be selected from the group consisting of Chu (N6) (Duchefa C0204), Gamborg B5 and MS medium, of which MS medium is preferably used. When using MS medium, it is preferable that 2, 4-D is included, and the concentration of 0.1-1.0 mg / L is more preferable, and the concentration of 0.2-0.5 mg / L is most preferable.
상기 단계 2에서 생성된 캘러스를 배양함으로써 단계 3의 배발생 세포가 형성된다.Embryonic cells of step 3 are formed by culturing the callus generated in step 2 above.
단계 4에 있어서, 배지는 상기 단계 2에서 사용된 것과 동일한 종류의 배지를 사용할 수 있으며, 이 중에서 MS 배지를 사용하는 것이 바람직하다.In step 4, the medium may be the same kind of medium as used in step 2 above, and it is preferable to use MS medium.
본 발명자들은 브로콜리의 체세포배 배양을 위하여, 바람직한 실시예로서 브로콜리 약 유래 배로부터 캘러스를 유도한 후 그로부터 체세포배를 생산하였다. 구체적으로, 브로콜리 약을 배양한 후 2O일 정도 경과된 시점부터 배(embryo)가 유기되었으며, 상기 배를 절단한 후 배지에 치상하여 배양(도 1 참조)한 결과, 잘린 배 조직이 비대해지고 잘린 부위에 캘러스가 형성되었다(도 2 참조). 이렇게 형성된 캘러스를 동일배지에 계대한 결과, 처음 형성된 캘러스는 하얗고 윤기가 없으며 조직이 엉성해 보였는데 수 차례 계대를 할수록 캘러스가 증식되면서 윤기가 나고 돌기를 형성하는 배발생 캘러스(embryogenic callus)가 형성되었다(도 3 참조). 상기 배발생 캘러스를 계대 후, 형성된 브로콜리 배발생 세포(embryogenic cells)를 액체배지가 들어있는 삼각플라스크에서 암소(dark condition)에서 현탁 배양하면(도 4 참조), 배발생 세포는 세포괴(cell cluster)를 형성하는데, 20일정도 배양하였을 때 최초 중량의 약 10배정도로 생중량이 증가한다. 이들 중 구상형배가 형성된 것들은 동일배지에 계대하여 형광빛 조건에서 배양하고, 나머지 세포는 새 배지에 옮겨 상기와 동일한 방법으로 배양함으로써 배발생세포는 배발생능을 유지하면서 증식된다(도 5 참조). 상기의 방법으로 형성된 식물체싹을 배양접시에서 배양하면 브로콜리의 유식물체가 형성된다(도 6 참조). 따라서, 본 발명자들은 상기와 같은 체세포배 배양 방법에 의하여 브로콜리의 배발생 세포가 영구적으로 유지 및 증식이 가능함을 확인하였다.The present inventors induced somatic embryos from broccoli drug-derived embryos as a preferred embodiment for somatic embryo culture of broccoli and then produced somatic embryos therefrom. Specifically, after culturing the broccoli drug was embryos (embryo) was organic from the time passed about 20 days, after cutting the embryos and cultured on the medium (see Figure 1 ), Cut embryo tissue was enlarged and callus was formed at the cut site (see FIG. 2 ). As a result of passing the callus formed on the same medium, the first callus formed was white, glossy, and had a loose texture. As the passage was repeated several times, an embryogenic callus was formed, which became shiny and formed as projections. (See FIG. 3 ). After passage of the embryogenic callus, the formed broccoli embryonic cells (embryogenic cells) in suspension incubated in a dark (dark condition) in a conical flask containing a liquid medium (see Fig. 4 ), embryogenic cells are cell clusters (cell cluster) When it is incubated for 20 days, the raw weight increases to about 10 times the initial weight. Among them, spherical embryos are cultured in the fluorescent medium under passage on the same medium, and the remaining cells are transferred to a new medium and cultured in the same manner as above, so that embryogenic cells are expanded while maintaining embryogenic capacity (see FIG. 5 ). . When the plant sprouts formed by the above method are cultured in a culture dish, seedlings of broccoli are formed (see FIG. 6 ). Accordingly, the present inventors confirmed that embryogenic cells of broccoli can be maintained and proliferated permanently by the somatic embryo culture method as described above.
또한, 본 발명은 상기 체세포배 배양 방법을 이용한 브로콜리 식물체싹의 대량 생산 방법을 제공한다.The present invention also provides a method for mass production of broccoli plant shoots using the somatic embryo culture method.
본 발명의 대량 생산 방법은Mass production method of the present invention
1) 배발생 세포(embryogenic cells)를 선발하여 배양하는 단계;1) selecting and culturing embryonic cells;
2) 단계 1의 세포를 증식시키고 형성된 세포괴를 분리하는 단계; 및2) proliferating the cells of step 1 and isolating the formed cell mass; And
3) 단계 2의 세포괴를 대량 배양하는 단계를 포함한다.3) mass culturing the cell mass of step 2;
단계 1에 있어서, 배발생 세포는 브로콜리의 종자, 약, 뿌리, 줄기 및 잎으로 구성된 군으로부터 선택된 조직으로부터 유래될 수 있으며, 이 중에서 브로콜리의 종자 또는 약으로부터 유래되는 것이 바람직하고, 브로콜리의 약으로부터 유래되는 것이 더욱 바람직하다.In step 1, the embryogenic cells may be derived from a tissue selected from the group consisting of seeds, drugs, roots, stems and leaves of broccoli, preferably from seeds or drugs of broccoli, and from the drug of broccoli It is more preferable to derive.
본 발명의 대량 생산 방법은 기존에 브로콜리의 종자를 사용하여 얻을 수밖에 없었던 브로콜리의 식물체싹을 배를 형성할 수 있는 브로콜리의 조직을 이용하여 체세포 배발생 방법으로 배양함으로써 간편하고 저렴하게 브로콜리의 식물체싹을 대량 생산할 수 있다.The mass production method of the present invention is a simple and inexpensive plant bud of broccoli by culturing in a somatic embryogenesis method using a broccoli tissue capable of forming a pear, which has been inevitably obtained using a seed of broccoli. Can be mass produced.
또한, 본 발명은 상기의 방법으로 제조된 브로콜리의 식물체싹을 포함하는 암의 예방 또는 치료용 건강식품을 제공한다.In another aspect, the present invention provides a health food for the prevention or treatment of cancer comprising a plant shoot of broccoli prepared by the above method.
앞서 언급한 바와 같이, 브로콜리의 식물체싹은 높은 농도의 설포라판을 함유하고 있다. 따라서, 본 발명의 암의 예방 또는 치료용 건강식품은 브로콜리의 식물체싹을 그대로 사용하거나 건조시킨 후 분말 형태로 사용할 수 있으며, 또한 다른 식품 또는 식품 성분과 함께 사용할 수 있으며 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분으로서 브로콜리 식물체싹의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있으며, 유효성분인 브로콜리 식물체싹은 안전성 면에서 아무런 문제가 없기 때문에 특정한 최대 허용 범위가 없이 사용될 수 있음은 확실하다.As mentioned earlier, the plant shoots of broccoli contain high concentrations of sulforaphane. Therefore, the health food for preventing or treating cancer of the present invention can be used as a powder form after using the plant bud of broccoli as it is or drying, and can also be used with other food or food ingredients and according to a conventional method as appropriate Can be used. The blending amount of broccoli plant buds as an active ingredient can be suitably determined according to its purpose of use (prevention, health or therapeutic treatment), and the active ingredient broccoli plant sprouts have no problem in terms of safety, so there is no specific maximum allowable range. It is certain that it can be used.
상기 건강식품의 종류에는 특별한 제한은 없다. 브로콜리 식물체싹을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 반드시 이에 한정되지 않음은 당업자에게 있어서 자명하다.There is no particular limitation on the kind of the health food. Examples of foods to which broccoli plant sprouts can be added include meat, sausages, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, teas and drinks. Alcoholic beverages and vitamin complexes, and the like, but are not necessarily limited thereto.
또한, 본 발명은 상기의 방법으로 제조된 브로콜리의 식물체싹을 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, including plant shoots of broccoli prepared by the above method.
본 발명의 약학적 조성물은 필요에 따라서 환제, 과립제, 정제 또는 캅셀제 형태로 제조할 수 있고, 필요에 따라 사용시에 다른 제형으로 바꾸어 사용할 수도 있다.The pharmaceutical composition of the present invention may be prepared in the form of pills, granules, tablets or capsules as needed, and may be used in other dosage forms when used.
본 발명의 약학적 조성물은 임상 투여시에 경구로 투여하는 것이 바람직하며 일반적인 의약품제제의 형태로 사용될 수 있다. 즉, 본 발명의 브로콜리의 식물체싹은 실제 임상 투여시에 경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 브로콜리의 식물체싹에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose) 또는 락토오스(Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 본 발명의 조성물은 환자의 체중, 연령, 성별, 병증의 경중정도와 소화상태에 따라 복용량을 증감할 수 있다.The pharmaceutical composition of the present invention is preferably administered orally during clinical administration and can be used in the form of general pharmaceutical preparations. That is, the plant shoots of broccoli of the present invention may be administered in various oral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, humectants, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like on plant buds of broccoli. (Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. The composition of the present invention can increase or decrease the dosage depending on the weight, age, sex, severity of the patient and digestive status.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 브로콜리의 체세포배발생(somatic embryogenesis)Example 1 Somatic Embryogenesis of Broccoli
본 발명자들은 브로콜리의 체세포배 배양을 위하여, 브로콜리 약에서 유래된 배로부터 캘러스를 유도한 후 그로부터 체세포배를 생산하였다. 구체적으로, 브로콜리 약배양을 하기 위하여 브로콜리의 꽃봉오리(bud)를 채취하여 70% 에탄올에 10초간, 2.5% 소디움 하이포클로라이트액(sodium hypochlorite solution)에 15분간 소독한 후 멸균수를 이용하여 수차례 세척하였다. 소독된 봉오리 중 1핵기 전후 시기의 소포자를 가지는 약을 채취하여 14%의 수크로즈(sucrose)(Duchefa S0809), 0.1 ㎎/ℓ NAA (Duchefa N0903), 0.1 ㎎/ℓ 2,4-D (Sigma D4517) 및 125 ㎎/ℓ AgNO3 (Duchefa S0536)를 포함하는 B5 수정배지(표 2 참조)(pH 5.8로 조정한 후 121℃, 1.2기압 하에서 습열멸균)가 들어있는 페드리디쉬에 치상한 후 32℃에서 24시간동안 고온처리하였으며, 이후 25℃에서 암배양하였다. 상기와 같이 배양 후 2O일이 경과된 시점부터 배가 유기되었다.We induced somatic embryos from broccoli drugs for culturing somatic embryos of broccoli and then producing somatic embryos therefrom. Specifically, in order to broccoli medicine culture buds of the broccoli (bud) is taken and sterilized in 70% ethanol for 10 seconds, 2.5% sodium hypochlorite solution for 15 minutes and then using sterile water It was washed once. From the sterilized buds, medicines with vesicles from around 1 nucleus were collected and 14% sucrose (Duchefa S0809), 0.1 mg / L NAA (Duchefa N0903), 0.1 mg / L 2,4-D (Sigma D4517) and B5 fertilization medium containing 125 mg / l AgNO 3 (Duchefa S0536) (see Table 2 ) (adjusted to pH 5.8 and then healed in fedridish containing 121 ° C, moist heat sterilization at 1.2 atmospheres). Heat treatment was performed at 32 ° C. for 24 hours, followed by dark culture at 25 ° C. As described above, embryos were organic from the time point of 20 days after culture.
상기 배로부터 캘러스를 유도하기 위하여, 배를 절단한 후 3% 수크로즈, 0.6% 식물성아가(phytoagar)(Duchefa P1003)를 포함하며, 0.2 ㎎/ℓ 또는 0.5 ㎎/ℓ 2,4-D가 포함된 MS 배지(pH 5.8)에 각각 치상하였다(도 1). 그 결과, 잘린 배 조직이 비대해지고 잘린 부위에 캘러스가 형성되었다(도 2). 상기 캘러스를 3∼4주 간격으로 동일 배지로 계대하였다.To induce callus from the embryo, the embryo was cut and then 3% sucrose, 0.6% phytoagar (Duchefa P1003), including 0.2 mg / L or 0.5 mg / L 2,4-D. Each was cultured in MS medium (pH 5.8) ( FIG. 1 ). As a result, the cut embryo tissue was enlarged and callus was formed at the cut region ( FIG. 2 ). The callus was passaged with the same medium at intervals of 3-4 weeks.
그 결과, 처음 형성된 캘러스는 하얗고 윤기가 없으며 조직이 엉성해 보였는데, 수차례 계대를 할수록 캘러스가 증식되면서 윤기가 나고 돌기를 형성하는 배발생 캘러스가 형성되었다(도 3).As a result, the initially formed callus was white, not glossy, and the tissues were coarse. As the passage was repeated several times, the callus was multiplied so that the embryogenic callus was formed to form luster and protrusion ( FIG. 3 ).
상기 배발생 캘러스로부터 생성된 브로콜리 배발생 세포를 1O ㎖의 호르몬이 첨가되지 않은 MS 액체배지가 들어있는 300 ㎖의 삼각플라스크에서 100 rpm, 25℃, 암소에서 현탁 배양하였다(도 4). 배발생 세포는 상기 배양조건에서 분열증식을 계속하면서 세포괴를 형성하는데, 20일정도 배양하였을 때 최초 중량의 약 10배정도로 생중량이 증가하였다. 이들 중 구상형배가 형성된 것들은 동일배지에 계대하여 형광빛 조건에서 배양하였다. 나머지 세포는 새 배지에 옮겨 상기와 동일한 방법으로 배양하였으며, 이렇게 함으로써 배발생 세포는 배 발생능을 유지하면서 증식되었다(도 5). 상기의 방법으로 형성된 식물체싹을 배양접시에서 배양하면 브로콜리의 유식물체가 형성되었다(도 6). 상기 실험결과를 하기 표 3에 요약하였다.Broccoli embryogenic cells produced from the embryogenic callus were suspended in 100 rpm, 25 ° C., cow in a 300 ml Erlenmeyer flask containing 10 ml of hormone-free MS liquid medium ( FIG. 4 ). Embryonic cells continue to divide and grow in the above culture conditions to form cell masses. After 20 days of culture, the embryonic cells increased to about 10 times the initial weight. Among them, globular embryos were cultured under fluorescent light under passage on the same medium. The remaining cells were transferred to fresh medium and cultured in the same manner as above, whereby embryonic cells proliferated while maintaining embryogenic capacity ( FIG. 5 ). Plant sprouts formed by the above method were cultured in a culture dish to form a seedling of broccoli ( FIG. 6 ). The experimental results are summarized in Table 3 below.
상기 표 3에서 알 수 있는 바와 같이, 브로콜리 유전자형(genotype) 별로 호르몬의 농도 차이는 있으나, 전체적으로 볼 때 생성된 캘러스를 계대함으로써 배(embryo)의 유기율이 좋아짐을 확인하였다. 즉, BA12는 2,4-D가 0.2 ㎎/ℓ인 경우에 동일배지에 계대를 할수록 배 유기정도가 눈에 띠게 증가하였으며, BB1의 경우에는 0.2 ㎎/ℓ 2,4-D와 0.5 ㎎/ℓ 2,4-D에서 모두 반응을 보였는데, 동일배지에 두차례 이상 계대한 후 배(embryo)가 가장 많이 유기됨을 볼 수 있다.As can be seen in Table 3 , there is a difference in hormone concentration for each broccoli genotype, but when viewed as a whole it was confirmed that the organic ratio of the embryo (embryo) is improved by passage. That is, BA12 significantly increased the degree of embryo induction with passage of the same medium in the case of 2,4-D of 0.2 mg / L, and 0.2 mg / L of 2,4-D and 0.5 mg / L of BB1. All reactions were performed at ℓ 2,4-D, and the embryos were the most organic after two passages in the same medium.
상기 결과로부터, 본 발명자들은 체세포배 배양 방법에 의하여 브로콜리의 배발생 세포가 영구적으로 유지 및 증식이 가능함을 확인하였다.From the above results, the inventors confirmed that embryogenic cells of broccoli can be permanently maintained and proliferated by the somatic embryo culture method.
상기에서 살펴본 바와 같이, 본 발명의 브로콜리의 체포 배 배양 방법은 종자로부터 얻을 수밖에 없었던 브로콜리의 식물체싹을 브로콜리의 체세포를 이용하여 대량 생산하게 함으로써 짧은 시간 내에 간편하게 무한정으로 브로콜리의 식물체싹을 생산할 수 있으며, 상기의 방법으로 개발된 브로콜리의 식물체싹은 항암식품 또는 의약품의 원료로써 값싸게 생산될 수 있을 뿐만 아니라, 브로콜리의 형질전환 등 생명공학기술에 매우 유용하게 사용될 수 있다. As described above, the method of culturing the arrested pear culture of broccoli of the present invention can produce broccoli plant buds indefinitely in a short time by allowing mass production of broccoli plant buds using broccoli somatic cells. The plant bud of broccoli developed by the above method can be produced inexpensively as a raw material of anticancer food or medicine, and can be very useful for biotechnology such as transformation of broccoli.
도 1은 약배양 방법을 통하여 형성된 배(embryo)를 이용하여 캘러스를 유도하는 사진이고, 1 is a photograph of inducing callus using an embryo (embryo) formed through the drug culture method,
도 2는 절단면으로부터 유기된 캘러스를 보여주는 사진이고, 2 is a photograph showing the callus induced from the cut surface,
도 3은 배발생 캘러스(embryogenic callus)가 유기된 것을 보여주는 사진이고, 3 is a photograph showing that the embryogenic callus (embryogenic callus) is organic,
도 4는 배발생세포(embryogenic cell)의 현탁배양을 보여주는 사진이고, Figure 4 is a photograph showing the suspension culture of embryogenic cells (embryogenic cells),
도 5는 브로콜리 식물체싹의 대량 생산 방법을 보여주는 사진이고, Figure 5 is a photograph showing a mass production method of broccoli plant shoots,
도 6은 체세포 배발생으로부터 유기된 유식물체(plantlet)를 보여주는 사진이다. Figure 6 is a photograph showing a plantlet (plantlet) derived from somatic embryogenesis.
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| KR970014605A (en) * | 1995-09-27 | 1997-04-28 | 킴 에스. 미첼 | Brassica vegetable supplements and preparation methods thereof |
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| KR20000058703A (en) * | 2000-06-26 | 2000-10-05 | 김이엽 | Method for mass propagation of Acanthopanax sessiliflorus(Rupr.et Max.) Seem. via somatic embryogenesis and mass production for medicinal and food material through large scale culture |
| KR20000066625A (en) * | 1999-04-19 | 2000-11-15 | 유청기 | Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture and Use Thereof |
| KR20010053342A (en) * | 1998-07-01 | 2001-06-25 | 존스 홉킨스 스쿨 오브 메디슨 | Development of novel, highly chemoprotectant crucifer germplasm |
| KR20020063914A (en) * | 1999-12-20 | 2002-08-05 | 코니스 프랑스, 에스.에이. | Cosmetic and/or pharmaceutical preparations |
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| KR970014605A (en) * | 1995-09-27 | 1997-04-28 | 킴 에스. 미첼 | Brassica vegetable supplements and preparation methods thereof |
| KR19990080042A (en) * | 1998-04-13 | 1999-11-05 | 석대은 | Functional drink containing broccoli vegetable and its manufacturing method |
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| KR19990036572A (en) * | 1998-07-23 | 1999-05-25 | 최용의 | Novel embryonic germ cells, somatic embryos and seedling production methods |
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| KR20020063914A (en) * | 1999-12-20 | 2002-08-05 | 코니스 프랑스, 에스.에이. | Cosmetic and/or pharmaceutical preparations |
| KR20000058703A (en) * | 2000-06-26 | 2000-10-05 | 김이엽 | Method for mass propagation of Acanthopanax sessiliflorus(Rupr.et Max.) Seem. via somatic embryogenesis and mass production for medicinal and food material through large scale culture |
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