KR20000066625A - Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture and Use Thereof - Google Patents
Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture and Use Thereof Download PDFInfo
- Publication number
- KR20000066625A KR20000066625A KR1019990013865A KR19990013865A KR20000066625A KR 20000066625 A KR20000066625 A KR 20000066625A KR 1019990013865 A KR1019990013865 A KR 1019990013865A KR 19990013865 A KR19990013865 A KR 19990013865A KR 20000066625 A KR20000066625 A KR 20000066625A
- Authority
- KR
- South Korea
- Prior art keywords
- ginseng
- cells
- embryogenic
- seedlings
- somatic embryos
- Prior art date
Links
- 235000008434 ginseng Nutrition 0.000 title claims abstract description 77
- 240000004371 Panax ginseng Species 0.000 title claims abstract description 65
- 230000000408 embryogenic effect Effects 0.000 title claims abstract description 52
- 230000000392 somatic effect Effects 0.000 title claims abstract description 31
- 235000002789 Panax ginseng Nutrition 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- 210000002257 embryonic structure Anatomy 0.000 title claims description 30
- 238000004114 suspension culture Methods 0.000 title claims description 10
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims abstract description 59
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 59
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 229920001817 Agar Polymers 0.000 claims abstract description 20
- 239000008272 agar Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 15
- 241000208340 Araliaceae Species 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 43
- 210000002308 embryonic cell Anatomy 0.000 claims description 23
- 239000002994 raw material Substances 0.000 claims description 7
- 241000196324 Embryophyta Species 0.000 claims description 6
- 239000005648 plant growth regulator Substances 0.000 claims description 6
- 244000269722 Thea sinensis Species 0.000 claims description 5
- 235000013361 beverage Nutrition 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 4
- 235000015872 dietary supplement Nutrition 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 230000030118 somatic embryogenesis Effects 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 2
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 claims 1
- 241000723346 Cinnamomum camphora Species 0.000 claims 1
- 229960000846 camphor Drugs 0.000 claims 1
- 229930008380 camphor Natural products 0.000 claims 1
- 239000000706 filtrate Substances 0.000 claims 1
- 239000005556 hormone Substances 0.000 claims 1
- 229940088597 hormone Drugs 0.000 claims 1
- 206010020649 Hyperkeratosis Diseases 0.000 abstract description 26
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 9
- 235000013402 health food Nutrition 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 230000001954 sterilising effect Effects 0.000 abstract description 2
- 239000000725 suspension Substances 0.000 abstract 2
- 239000000825 pharmaceutical preparation Substances 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000003375 plant hormone Substances 0.000 description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000018997 Growth Hormone Human genes 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 241000220324 Pyrus Species 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- 239000003630 growth substance Substances 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- JZUFKLXOESDKRF-UHFFFAOYSA-N Chlorothiazide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC2=C1NCNS2(=O)=O JZUFKLXOESDKRF-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- -1 ODS Substances 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000490 cosmetic additive Substances 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229940107131 ginseng root Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000013930 proline Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000014393 valine Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/16—Tea extraction; Tea extracts; Treating tea extract; Making instant tea
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
- A23V2250/2124—Ginseng
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Environmental Sciences (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Sustainable Development (AREA)
- Birds (AREA)
- Alternative & Traditional Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Cosmetics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
본 발명은 인삼(Panax ginseng C.A. Meyer - 분류학상 장뇌삼 및 산삼 포함)의 배발생세포(Panax ginseng / Embryogenic cells)를 액체배지에서 배양하여 체세포배(somatic embryo) 및 유식물체(seedling 또는 plantlet)의 대량 생산 방법을 세계 최초로 개발하여 이들을 생식용 또는 식품, 음료, 차, 화장품 및 신기능성 건강보조식품 등의 가공용 재료 및 유용성분의 추출용 재료로 사용하는 것과 유식물체를 순화시켜 재배용 묘삼으로 사용하도록 공급하는데 있다.The present invention is cultured in a liquid medium of Panax ginseng CA Meyer (Panax ginseng / Embryogenic cells) of ginseng (Panax ginseng CA Meyer-including taxonomy ginseng) and a large amount of somatic embryo (seedling or plantlet) Developed the world's first production method and used them as raw materials for processing or extracting useful ingredients such as food, beverages, tea, cosmetics and new functional health supplements, and purified seedlings for use as cultivated seedlings It is.
인삼은 3월에 싹이 나오며, 5월 중순에 꽃이 피는 오갈피나무과(Araliaceae)의 다년생 초본으로 40-60 cm 가량 곧게 자란다. 우리 나라 인삼은 세계적으로 고려인삼(Korean Ginseng)으로 널리 알려져 약효가 좋아서 영약(靈藥)이라고 부르고 있다. 인삼은 원래 야생이었으나 재배법의 발달로 노지에서 재배하게 되었는데 야생의 것을 산삼 또는 야산삼이라 부르고, 산삼의 종자를 채취하여 밭에서 야생의 상태로 재배한 것을 장뇌(또는 長蘆)라하며, 예로부터 집에서 재배하는 것을 가삼(家蔘), 인삼(人蔘), 원삼(園蔘)이라 부른다(원색천연약물대사전, 남산당, 1984).Ginseng sprouts in March, and is a perennial herb of the Araliaceae, which blooms in mid-May, growing up to 40-60 cm. Korean ginseng is widely known as Korean Ginseng in the world. Ginseng was originally wild, but it was cultivated in open field due to the development of cultivation methods. Wild ginseng is called wild ginseng or wild ginseng, and the seeds of wild ginseng are harvested and grown in the wild in the field. Cultivation at home is called ginseng, ginseng and Wonsam (Natural natural drug metabolism, Namsandang, 1984).
인삼은 맛이 약간 달고 쓰며 성질은 따뜻하다고 알려져 있으며, 인삼을 먹으면 정신적, 육체적 활동력이 강화되고 피로가 빨리 회복되고, 정신을 안정시키며 눈을 밝게 하고 기억력을 좋게 한다고 한다. 또한 장기간 복용하면 장수(동의고전)하며, 강장작용은 인삼의 잎, 줄기, 꽃, 열매에도 있는 것으로 알려져 있다. 인삼은 면역 글로블린의 양과 임파세포수를 늘리고 임파세포의 유약화를 촉진시키며, 망상내피계통의 기능을 강화할 뿐 아니라 몸에 나쁜 영향을 주는 물리적 화학적 요인에 대한 저항성(약용식물의 이용과 신재배기술, 선진문화사, 1992)을 높이는 것으로 보고되어 있다.Ginseng is known to be slightly sweet and bitter and warm in nature, and it is said that eating ginseng strengthens mental and physical activity, quickly recovers fatigue, stabilizes the mind, brightens the eyes and improves memory. In addition, if taken for a long time (long-term agreement), the tonic effect is known to the leaves, stems, flowers, fruits of ginseng. Ginseng increases the amount of immune globulin and the number of lymphocytes, promotes lymphatic cell weakening, strengthens the function of the reticuloendothelial system, as well as resistance to physical and chemical factors that adversely affect the body (the use of medicinal plants, new cultivation techniques, Advanced Culture History, 1992).
인삼에는 배당체(여러 가지 인삼지드와 다우코스테린), 향기름(파난첸), 아미노산(글루타민산, 발린, 프롤린, 알라닌, 아르기닌 등), 비타민(B1, B2, C, 니코틴산, 판토텐산 등), 유기산(팔미틴산, 스테아린산, 올레인산, 리놀산 등), 탄수화물(포도당, 과당, 사탕, 맥아당, 녹말, 펙틴 등) 등을 함유하고 있다.Ginseng contains glycosides (various ginseng zide and doukosterine), fragrance (pananchen), amino acids (glutamic acid, valine, proline, alanine, arginine, etc.), vitamins (B 1 , B 2 , C, nicotinic acid, pantothenic acid, etc.) Organic acids (palmitinic acid, stearic acid, oleic acid, linoleic acid, etc.), carbohydrates (glucose, fructose, candy, maltose, starch, pectin and the like).
인삼은 농가에서 재배할 경우 종자의 채종, 개갑처리, 묘포관리 등에 어려움이 많다. 또한, 인삼의 노지재배시 농약오염, 환경파괴 등의 문제점과 많은 인력 및 경비가 소요된다. 따라서 최근에는 인삼을 조직배양하여 세포, 부정근, 모상근 등을 대량으로 생산하려는 연구가 진행 중에 있다. 실제 일본의 日東電工사는 인삼 세포를 용기 내에서 대량으로 배양하여 사포닌을 추출하는데 성공하였고, 그 배양체를 특별히 가공하지 않고 그대로 건조, 분말화하여 건강식품으로 시판하고 있다. 국내에서도 인삼의 자엽절편을 한천배지에서 배양하여 체세포배를 유도하여 재분화시키는 연구(특허출원번호 1997-015999)와 인삼의 모상근으로부터 배형성캘러스(또는 배발생캘러스)를 한천배지에서 유도한 후 이들로부터 체세포배발생과정을 거처 유식물체를 얻어 토양에 직접 이식하는 방법이 보고되었다(특허출원번호 1997-016000). 또한, 인삼의 부정근 또는 모상근배양을 대량으로 생산하여 유용성분추출용으로 사용하고자 연구를 진행하고 있다(임업연구원, 인삼연초연구원). 그러나 아직까지 액체배지에서 세포를 배양하여 체세포배 및 유식물체의 대량생산 방법은 보고되지 않았다. 이는 인삼의 특징상 한천배지에서 배발생캘러스는 잘 유도되어 이들로부터 채세포배를 얻는 것은 어렵지 않지만, 배발생캘러스를 액체배지에서 배양하였을 때 쉽게 세포화되지 않는다. 간혹 액체배지에서 배발생캘러스가 세포화되어 분열 증식이 되는 경우가 있지만 이들은 배발생능력이 없이 단지 세포 분열만 일어나 아직까지 액체배지에서 배발생능력을 가진 배발생세포를 만들지 못하고 있으므로 액체배지에서 세포배양에 의한 대량의 체세포배 유도는 이루어지지 못하고 있다. 인삼과 같이 경작에 의한 재배가 까다로운 식물의 경우 조직배양에 의해 배발생세포, 체세포배, 유식물체를 대량으로 증식시켜 재배 인삼의 대용으로 사용한다면 노동력과 경비 및 시간 등을 획기적으로 줄일 수 있어 엄청난 경제적 파급효과가 있을 것이다.When ginseng is grown in farms, it is difficult to collect seeds, process gag, and manage seedlings. In addition, when cultivating the ginseng field, it takes a lot of problems such as pesticide pollution and environmental destruction, and manpower and expense. Therefore, in recent years, research has been conducted to produce large amounts of cells, roots, hairy roots and the like by tissue culture of ginseng. In fact, Japan's Nippon Denki Co., Ltd. has succeeded in extracting saponin by cultivating ginseng cells in a large amount in a container, and drying and powdering the culture as it is without processing it as a health food. In Korea, cultivation of cotyledon slices of ginseng in agar medium to induce somatic embryos (Patent Application No. 1997-015999) and after induction of embryogenic callus (or embryogenic callus) from hairy root of ginseng in agar medium A method of obtaining seedlings and transplanting them directly into soil after somatic embryogenesis has been reported (Patent Application No. 1997-016000). In addition, research is underway to produce a large amount of ginseng root or hairy root culture of ginseng for use in extracting useful components (Forestry Research Institute, Ginseng and Tobacco Research Institute). However, there have been no reports on mass production of somatic embryos and seedlings by culturing cells in liquid medium. It is not difficult to obtain embryonic callus in agar medium because of the characteristics of ginseng, and it is not difficult to obtain cell embryos from them, but when the embryogenic callus is cultured in a liquid medium, it is not easily cellized. Occasionally, the embryogenic callus is cellized in the liquid medium to divide and proliferate. However, these cells do not have an embryogenic capacity and only cell division occurs. Thus, cells in the liquid medium have not yet produced embryonic cells with embryogenic capacity. Induction of a large amount of somatic embryos by culture has not been achieved. For plants that are difficult to cultivate by cultivation, such as ginseng, it is possible to drastically reduce labor, expense, and time by using large amounts of embryonic cells, somatic embryos, and seedlings by using tissue culture to replace cultivated ginseng. There will be an economic ripple effect.
따라서 본 발명자들은 건강식품 및 유용성분 추출용 재료로 이용 가능한 인삼의 배발생세포, 체세포배 또는 유식물체를 대량으로 얻기 위해 액체배지에서 인삼의 배발생능력을 가진 배발생세포를 만들어 세포배양에 의한 대량의 체세포배 및 유식물체를 얻는 배양법을 개발하였다. 한천배지에서 배발생캘러스를 유도하여 약 4주 간격으로 2∼3회 계대배양하면 배발생능을 가진 부서지기 쉬운 배발생캘러스가 적은 빈도로 유도되는데 이들을 식물생장조절물질이 첨가되지 않은 MS 액체배지에 옮겨 약 3주간 현탁배양하면 배발생세포가 증식하는 동시에 배발생세포괴도 생성된다. 그물망(크기 250㎛)으로 걸러 망을 통과한 배발생세포는 재차 세포분열 및 증식용으로 사용하고, 그물망을 통과하지 못한 배발생세포괴는 생장조절물질이 첨가되지 않은 MS 액체배지에서 약 3주마다 계대배양하면 구상형배, 심장형배, 어뢰형배 및 성숙한 체세포배(자엽기배)가 된다. 결국 이들 자엽기배는 뿌리가 신장하여 유식물체로 성장하게 된다. 이러한 배양법에 의해 배발생세포 및 체세포배를 대량으로 얻어서 이들을 약용 성분의 추출용 재료 및 건강식품으로의 활용이 가능하고, 체세포배 발생과정을 거처 얻어진 다량의 유식물체는 생식용 제품 또는 건강식품의 원료 및 유용물질의 추출용 재료로 사용할 수 있다. 또한, 유식물체를 활용하여 재배용 묘삼으로 사용하도록 공급할 수 있다.Therefore, the present inventors made embryonic cells having the embryogenic capacity of ginseng in liquid medium to obtain a large amount of embryonic cells, somatic embryos or seedlings of ginseng that can be used as a material for extracting health foods and useful ingredients. Culture methods have been developed to obtain large somatic embryos and seedlings. Induced embryonic callus in agar medium and subcultured two to three times at intervals of about 4 weeks, the fragile embryogenic callus with embryogenic ability is induced with a low frequency, which is MS liquid medium without plant growth regulator added. Suspension cultures for about three weeks after the transfer to embryonic embryonic cells proliferate and generate embryogenic cell mass. Embryonic cells that have passed through the net (250 μm in size) are used for cell division and proliferation again. Embryonic cell masses that do not pass through the net are reapplied every 3 weeks in MS liquid medium without growth regulators. Passage cultures are globular, heart, torpedo and mature somatic embryos. Eventually these cotyledon cultivation grows to seedling roots. This culture method can be used to obtain embryonic embryonic cells and somatic embryos in large quantities, and to use them as extracting materials and health foods of medicinal components. It can be used as extraction material for raw materials and useful materials. In addition, it can be supplied for use as seedlings for cultivation by utilizing the seedlings.
본 발명은 인삼의 배발생세포(Panax ginseng / Embryogenic cells)를 식물호르몬이 첨가되지 않은 MS 액체배지에 현탁배양하여 증식시킨 후 250㎛ 그물망으로 걸러 망을 통과한 세포는 새배지에 다시 배양하여 배발생세포의 분열 및 증식용으로 사용하고, 망을 통과하지 못한 세포괴도 식물호르몬이 첨가되지 않은 MS 액체배지에서 약 3주 간격으로 계대배양하여 체세포배발생과정을 통해 대량의 유식물체를 얻어, 이를 한천배지에 이식하여 유식물체를 무균상태의 생식용 식품으로 상품화한다.The present invention suspends the growth of ginseng embryonic cells (Panax ginseng / Embryogenic cells) in MS liquid medium not added to the plant hormone, and then multiply through the 250㎛ mesh to pass through the network cultured in a new medium It is used for division and proliferation of embryonic cells, and cell cultures that do not pass through the network are passaged at intervals of about 3 weeks in MS liquid medium to which plant hormone is not added. Thus, a large number of seedlings are obtained through the somatic embryogenesis process. The seedlings are transplanted into agar medium and commercialized as sterile food for reproduction.
본 발명의 또 다른 목적은 현탁배양으로 대량생산한 인삼의 배발생세포, 체세포배 및 유식물체로부터 유용성분을 추출하여 식품, 음료, 화장품, 신기능성 의약품, 차의 원료 및 건강보조식품 등의 원료로 제공하는데 있다. 또한, 유식물체를 인공종자로 활용하여 외부환경에 순화시켜 재배용 묘삼으로 사용하도록 공급할 수 있는 것을 제시함으로서 본 발명을 완성하였다.Another object of the present invention is to extract useful ingredients from embryonic cells, somatic embryos and seedlings of ginseng mass-produced in suspension culture, raw materials such as food, beverages, cosmetics, new functional medicines, tea raw materials and health supplements To provide. In addition, the present invention has been completed by suggesting that the seedlings can be supplied to be used as cultivated seedlings by purifying the external environment using artificial seed.
이하 본 발명의 구성 및 작용을 상세히 설명하고자 한다.Hereinafter will be described in detail the configuration and operation of the present invention.
도 1a는 한천배지에서 유도된 인삼의 부서지기 쉬운 배발생캘러스(friable embryogenic callus)를 나타낸 사진이다.Figure 1a is a photograph showing the friable embryogenic callus of the ginseng derived from agar medium (friable embryogenic callus).
도 1b는 인삼의 부서지기 쉬운 배발생캘러스를 MS 액체배지에 옮겨 현탁배양하여 얻은 배발생세포(Panax ginseng / Embryogenic cells)를 나타낸 사진이다.Figure 1b is a photograph showing the embryonic cells (Panax ginseng / Embryogenic cells) obtained by suspending culture by transferring the fragile embryogenic callus of ginseng to MS liquid medium.
도 1c는 배발생세포괴를 식물생장 호르몬이 첨가되지 않은 MS 액체배지에서 배양하여 대량으로 체세포배를 유도한 것을 나타낸 사진이다.Figure 1c is a photograph showing the induction of somatic embryos in large quantities by culturing embryogenic cell mass in MS liquid medium without the addition of plant growth hormone.
도 1d는 성숙한 체세포배로부터 얻어진 인삼의 유식물체를 나타낸 사진이다.Figure 1d is a photograph showing the seedlings of ginseng obtained from mature somatic embryos.
본 발명은 개갑 처리된 인삼종자의 두꺼운 외껍질을 제거하고 배유를 멸균한 후, 배유내부의 배를 적출하여 자엽 또는 배축을 2 mm 정도의 길이로 절단하여 0.5∼1.0 mg/L 2,4-D가 첨가된 MS 한천배지에서 배양하여 단단한 배발생캘러스 (compact embryogenic callus)를 얻어 이들을 0.5∼1.0 mg/L 2,4-D가 첨가된 MS 한천배지에서 약 4주 간격으로 2∼3회 계대배양함으로써 단단한 배발생캘러스의 일부가 옅은 노랑색의 부서지기 쉬운 배발생캘러스(friable embryogenic callus)가 되었다. 이들은 식물호르몬이 첨가되지 않은 MS 액체배지에 배양하였을 때 배발생능을 가진 단세포 및 소세포군 상태의 배발생세포가 되었다. 상기 인삼의 배발생세포를 배양하여 증식시킨 후 250㎛ 크기의 멸균된 그물망으로 걸러 배양초기의 세포농도와 동일하게 하여 새 배지에서 배양하여 배발생능을 가진 세포를 다시 증식시킬 수 있다. 한편, 그물망을 통과하지 못한 세포괴는 식물생장호르몬이 첨가되지 않은 MS 액체배지에 계대배양하여 균일한 크기의 구상형, 심장형, 어뢰형 시기의 체세포 배발생과정을 거처 대량의 유식물체를 얻을 수 있다The present invention is to remove the thick outer shell of ginseng seed ginseng seeding treated and sterilized the endosperm, cut out the pear inside the endosperm to cut the cotyledon or hypocotyl to a length of about 2 mm to 0.5 ~ 1.0 mg / L 2,4- Cultured in D-added MS agar medium to obtain compact embryogenic callus, which were passaged 2-3 times at intervals of about 4 weeks in MS agar medium containing 0.5-1.0 mg / L 2,4-D. By culturing, a part of the hard embryogenic callus became a pale yellow friable embryogenic callus. When cultured in MS liquid medium to which no plant hormone was added, they became embryonic cells in the state of single cell and small cell group with embryogenic capacity. After culturing the embryonic embryonic cells of ginseng and cultivating the filter with a sterile net of 250㎛ size in the same manner as the initial concentration of the culture can be cultured in a new medium to re-grow the cells with embryogenic capacity. On the other hand, cell masses that do not pass through the net are passaged in MS liquid medium to which plant growth hormone is not added, so that a large number of seedlings can be obtained through the somatic embryogenesis of homogeneous globular, heart and torpedo types.
이하 본 발명을 다음의 실시 예를 통하여 보다 상세히 설명하고자 한다. 그러나 이들이 본 발명의 기술적 범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these do not limit the technical scope of the present invention.
＜실시예 1＞ 인삼 배발생캘러스로부터 배발생세포(Panax ginseng / Embryogenic cells)의 선발 및 현탁배양<Example 1> Selection and suspension culture of Panax ginseng / Embryogenic cells from ginseng embryogenic callus
개갑 처리된 인삼종자의 배유를 70% 에탄올로 30초간 1차 멸균한 후, 2% 차염소산나트륨으로 15분간 2차 멸균하였다. 멸균된 배유는 멸균수로 3회 세척한 후 물기를 제거하고, 배유내부의 배를 적출하여 자엽 및 배축을 각각 2 mm 정도의 절편으로 절단하여 0.5∼1.0 mg/L 2,4-D가 첨가된 MS (Murashige and Skoog) 한천배지(1.0% 한천, 3% 설탕, pH 5.8로 조정하여 121℃, 1.2 기압하에서 습열 멸균)에 이식하여 암소(24±1℃)에서 배양하였다. 멸균된 페트리디쉬(87 × 15 mm)에 배지를 25 ㎖씩 분주하고, 페트리디쉬당 10 절편씩을 치상하였다. 배양 4주 후 인삼의 자엽 및 배축 절편조직으로부터 단단한 배발생캘러스(compact embryogenic callus)가 유도되었다. 배발생캘러스를 선별하여 0.5-1.0 mg/L 2,4-D가 함유된 MS 한천배지에서 약 4주 간격으로 2∼3회 계대배양하였을 때 세포상태로 쉽게 유리될 수 있는 부서지기 쉬운 옅은 노랑색의 배발생캘러스(friable embryogenic callus)가 배지와 단단한 캘러스의 접촉면에서 유도되는 것이 드물게 관찰되었다. 이 배발생캘러스만을 선별하여 0.5∼1.0 mg/L 2,4-D가 함유된 MS 한천배지에 옮겨 암소에서 배양하였을 때 단단한 배발생캘러스와 구별되는 부서지기 쉬운 배발생캘러스의 형태로 증식되었다(도 1a). 부서지기 쉬운 배발생캘러스를 생장조절물질이 첨가되지 않은 200 mL MS 액체배지에 생중량 5 g 정도 넣어 밀도를 높게 하여 3주간 배양하였을 때 단세포 및 소세포군의 배발생세포를 대량으로 얻을 수 있다(도 1b). 본 발명의 인삼 배발생세포(Panax ginseng / Embryogenic cells)는 한국과학기술원 생명공학연구소내 유전자은행에 1999년 4월 14일 기탁번호 KCTC 0601BP로 기탁하였다. 증식된 배발생세포는 250㎛ 그물망으로 걸러 그물망을 통과한 세포는 새 배지에 옮겨 전과 동일한 방법으로 배양하였을 때 배발생세포는 계속 증식시킬 수 있었다. 한편, 그물망을 통과하지 못한 배발생세포괴는 식물생장조절물질이 첨가되지 않은 MS 액체배지에 옮겨 형광빛 조건에서 배양하여 체세포배를 유도하였다.The endosperm of the ginseng seed treated with Gaegi was first sterilized with 70% ethanol for 30 seconds and then sterilized with 2% sodium hypochlorite for 15 minutes. Sterilized endosperm is washed three times with sterile water and then drained. Extracted pears in endosperm are cut into cotyledons and hypocotyls in 2 mm sections and 0.5 to 1.0 mg / L 2,4-D is added. MS (Murashige and Skoog) agar medium (1.0% agar, 3% sugar, pH 5.8, adjusted to pH 5.8 moist heat sterilization at 121 ℃, 1.2 atm) was incubated in the cow (24 ± 1 ℃). 25 ml of the medium was dispensed into sterile Petri dishes (87 x 15 mm) and 10 sections per Petri dish were toothed. After 4 weeks of culture, hard embryogenic callus was induced from the cotyledons and hypocotyl sections of ginseng. Embryonic callus was screened and pale yellow that could be easily released into the cell state when passaged 2-3 times at intervals of about 4 weeks in MS agar medium containing 0.5-1.0 mg / L 2,4-D. It was rarely observed that the friable embryogenic callus was induced at the contact surface of the medium with the hard callus. The embryogenic callus alone was selected and transferred to MS agar medium containing 0.5-1.0 mg / L 2,4-D and grown in the form of a brittle embryogenic callus distinguished from hard embryogenic callus when cultured in cows. 1a). When the embryonic callus, which is fragile, is grown in 200 mL MS liquid medium without growth regulator, the density is increased to about 5 g, and cultured for 3 weeks, the embryonic cells of single cells and small cell groups can be obtained in large quantities. 1b). Panax ginseng (Embryogenic cells) of the present invention was deposited on April 14, 1999 in the gene bank of the Institute of Biotechnology, Korea Advanced Institute of Science and Technology (KCTC 0601BP). The expanded embryonic cells were filtered through a 250 μm mesh, and the cells passed through the mesh were transferred to a new medium and cultured in the same manner as before. The embryonic cells could continue to grow. On the other hand, embryogenic cell mass that failed to pass through the net was transferred to MS liquid medium to which no plant growth regulator was added, and cultured in fluorescent light conditions to induce somatic embryos.
＜실시예 2＞ 인삼 배발생세포(Panax ginseng / Embryogenic cells)로부터 대량의 체세포배 및 유식물체 생산<Example 2> Mass production of somatic embryos and seedlings from Panax ginseng / Embryogenic cells
인삼의 배발생세포(또는 세포괴)를 식물생장조절물질이 첨가되지 않은 MS 액체배지가 들어 있는 2∼5 ℓ의 용기에 옮겨 형광빛 조건에서 약 3주간 배양하면 구상형배가 유도된다. 처음 구상형배가 발달할 때에는 세포괴와 구상형배가 혼재되어 있는데 이들을 동일한 시기의 것으로 분리하기 위해 배양용기를 30초간 움직이지 않게 정치한 후 구상형배가 대부분 가라앉으면 천천히 다른 용기에 배지를 따라낸다. 이때 배지와 함께 먼저 옮겨지는 것은 무게가 가벼운 세포괴이고, 늦게까지 남는 것은 세포괴보다 무거운 구상형배가 배부분이다. 이와 같이 중력을 이용한 방법으로 계대배양해 주면 체세포배 발생과정에서 비교적 균일한 체세포배의 집단을 얻을 수 있다(도 1c). 구상배로부터 심장형배, 어뢰형배 및 자엽기 배의 발달은 약 3주 간격으로 새 배지에 계대배양함으로서 쉽게 이루어졌다. 체세포배가 성숙(자엽기 이후)함에 따라 계대배양 기간을 3주에서 2주 정도로 단축하였을 때 유식물체 또는 소식물체(seedlings or plantlets)의 형태가 양호하였다. 액체배지에서 유도된 유식물체는 식물생장조절물질이 첨가되지 않은 MS 한천배지로 옮겨 형광빛 아래에서 배양하였을 때 자엽과 뿌리가 정상적으로 발달한 균일한 유식물체가 되었다(도 1d).When ginseng embryonic cells (or cell masses) are transferred to a 2-5 L container containing MS liquid medium to which no plant growth regulators are added, they are cultured for about 3 weeks in fluorescent light conditions to induce globular embryos. When the first globular embryo develops, cell masses and globular embryos are mixed, and in order to separate them into the same period, the culture vessel is left still for 30 seconds, and when the globular embryo subsides, the medium is slowly poured into another container. At this time, the first to be transferred with the medium is light weight cell mass, and the late one is a globular embryo that is heavier than the cell mass. As such, when subcultured by the method using gravity, a population of relatively uniform somatic embryos can be obtained in the process of generating somatic embryos (FIG. 1C). The development of heart, torpedo and cotyledon embryos from globular embryos was easily accomplished by passage to fresh medium at about three week intervals. As the somatic embryo matured (after cotyledon), the passage of seedlings or plantlets was good when the passage period was shortened from 3 to 2 weeks. Seedlings derived from the liquid medium were transferred to MS agar medium without the addition of plant growth regulators and became a homogenous seedling with normal cotyledons and roots when cultured under fluorescent light (FIG. 1D).
＜실시예 3＞ 인삼 배발생세포(Panax ginseng / Embryogenic cells)로부터 대량 생산된 체세포배 및 유식물체의 사용 방법 및 용도<Example 3> Method and use of somatic embryos and seedlings produced in large quantities from Panax ginseng / Embryogenic cells
체세포배발생과정의 계대배양시 대량으로 유도된 체세포배는 회수하여 자연 건조시켜 분말 또는 건조 상태로 차 및 건강음료의 첨가제로 사용할 수가 있다. 또한, 액체배지에서 유식물체를 바로 회수하여 메탄올 또는 에탄올로 추출하고, 핵산, 에칠아세테이트, 부탄올, 및 물을 사용하여 순차적으로 분획한 분획물들을 실리카겔, ODS, 세파덱스 및 이온컬럼 크로마토그래피로 분획하고 분리하여 추출물 또는 분획물을 얻어 이들을 화장품, 건강식품, 의약제제 등의 조성물의 원료로 사용할 수 있다. 액체배지에서 유도된 유식물체는 식물생장조절물질이 첨가되지 않은 MS 한천배지로 옮겨 형광빛 아래에서 배양하여 자엽과 뿌리가 균일하게 발달한 유식물체로 배양하여 무균, 무공해의 생식용 인삼으로 사용할 수 있다.During passage of somatic embryo development, a large amount of somatic embryos can be recovered and dried naturally and used as additives for tea and health beverages in powder or dried form. In addition, the seedlings are directly recovered from the liquid medium and extracted with methanol or ethanol, and the fractions sequentially separated using nucleic acid, ethyl acetate, butanol, and water are separated by silica gel, ODS, Sephadex, and ion column chromatography. Separately, extracts or fractions can be obtained and used as raw materials for compositions such as cosmetics, health foods and pharmaceuticals. Seedlings derived from liquid medium can be transferred to MS agar medium without added plant growth regulators and cultured under fluorescent light to grow as a seedling plant with cotyledons and roots with uniform development. have.
또한, 한천배지에서 자엽과 뿌리가 정상적으로 발달한 유식물체는 핀셋으로 집어내어 배지가 묻지 않도록 물로 씻어준 후, 약토와 원야토를 1:3로 섞어만든 상토에 심어 습도가 항상 70∼80% 정도 유지되도록 한다. 상토에 이식한 유식물체는 뿌리가 활착될때까지 직사광선을 피하고 차양된 배양실이나 온실에서 기온 20∼25℃ 조건으로 키운다. 뿌리가 활착된 인삼은 포장에 옮겨 심는 묘삼으로 사용하여, 수확하고자하는 목적(홍삼용 또는 백삼용 등)에 맞게 적절한 갯수를 포장에 옮겨 심는다.In addition, seedlings with cotyledons and roots normally developed on agar plate should be picked up with tweezers and washed with water to prevent medium from sticking. To be maintained. Seedlings transplanted to the topsoil are kept under the condition of 20-25 ℃ in a shaded culture room or greenhouse until the roots stick. Ginseng rooted in roots is used as seedlings that are transferred to the packaging, and the appropriate number of seedlings for red ginseng or white ginseng is harvested.
본 발명은 인삼의 배발생세포를 현탁배양으로 짧은 기간내에 대량으로 증식시켜 간편하게 인삼 체세포배 및 유식물체를 대량으로 생산할 수 있는 방법을 제공한다. 이 유식물체는 MS 한천배지가 들어있는 멸균된 페트리디쉬에서 자엽과 뿌리가 정상적으로 발달한 균일한 유식물체로 성장시켜 무균, 무공해의 생식용 인삼으로 상품화할 수 있다. 또한, 유식물체를 순화시켜 재배용 묘삼으로 사용하도록 공급할 수 있다.The present invention provides a method that can easily produce a large amount of ginseng somatic embryos and seedlings by proliferating a large amount of embryonic development cells of ginseng in a suspension culture in a short period of time. The seedlings can be grown into sterile petri dishes containing MS agar medium and grown into uniform seedlings with normal cotyledons and roots and commercialized as sterile, pollution-free reproductive ginseng. In addition, the seedlings can be purified and supplied for use as cultivated seedlings.
본 발명의 또 다른 효과는 현탁배양으로 대량생산된 인삼의 체세포배 및 유식물체로부터 유용성분을 추출하여 식품, 음료, 건강보조식품 및 화장품의 첨가물 이외에도 차의 원료 및 의약품 등에 유용하게 사용될 수 있다.Another effect of the present invention is to extract useful components from the somatic embryos and seedlings of ginseng mass-produced in suspension culture, which can be usefully used for tea raw materials and pharmaceuticals in addition to food, beverages, dietary supplements and cosmetic additives.
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-1999-0013865A KR100367104B1 (en) | 1999-04-19 | 1999-04-19 | Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-1999-0013865A KR100367104B1 (en) | 1999-04-19 | 1999-04-19 | Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20000066625A true KR20000066625A (en) | 2000-11-15 |
| KR100367104B1 KR100367104B1 (en) | 2003-01-15 |
Family
ID=19580877
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-1999-0013865A KR100367104B1 (en) | 1999-04-19 | 1999-04-19 | Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100367104B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100398749B1 (en) * | 2000-09-15 | 2003-09-19 | (주)파낙시아 | Method for mass propagation of Wild Korean ginseng (Panax ginseng C.A.Meyer) by biotechnological technique |
| KR100497717B1 (en) * | 2002-10-29 | 2005-06-28 | 동부한농화학 주식회사 | Method of culturing somatic embryo of broccoli and method for the mass-production of broccoli sprouts using the same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05244838A (en) * | 1992-03-05 | 1993-09-24 | Mitsubishi Agricult Mach Co Ltd | Method for inducing adventitious embryo of medicinal ginseng |
| JPH0654631A (en) * | 1992-08-05 | 1994-03-01 | Mitsubishi Agricult Mach Co Ltd | Method for inducing adventitious embryo of ginseng |
| KR100241178B1 (en) * | 1997-04-28 | 2000-02-01 | 박명규 | The method of propagation hairy root of panax ginseng |
| KR100318090B1 (en) * | 1998-10-19 | 2002-06-20 | 대한민국(관리청:특허청장, 승계청:산림청 임업연구원장) | Mass production method of Sangubu-geunjeong (segeun) in Hansan using bioreactor |
| KR100333559B1 (en) * | 1999-02-22 | 2002-04-24 | 윤의수 | Novel process for mass production of embryogenic cells and seedling thereof on hormone independent medium in ginseng |
-
1999
- 1999-04-19 KR KR10-1999-0013865A patent/KR100367104B1/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100398749B1 (en) * | 2000-09-15 | 2003-09-19 | (주)파낙시아 | Method for mass propagation of Wild Korean ginseng (Panax ginseng C.A.Meyer) by biotechnological technique |
| KR100497717B1 (en) * | 2002-10-29 | 2005-06-28 | 동부한농화학 주식회사 | Method of culturing somatic embryo of broccoli and method for the mass-production of broccoli sprouts using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100367104B1 (en) | 2003-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Endress et al. | Plant cell biotechnology | |
| CN104273028B (en) | Method for rapid in-vitro propagation of Crassulaceae plant | |
| KR101938835B1 (en) | Method for vegetative propagation of Camellia japonica | |
| Ray et al. | In vitro regeneration of brinjal (Solanum melongena L.) | |
| Faisal et al. | In vitro plant regeneration from alginate-encapsulated microcuttings of Rauvolfia tetraphylla L | |
| CN101218896A (en) | Method for producing momordica grosvenori group seedling with single culture medium | |
| Yadav et al. | Micropropagation of Spilanthes acmella Murr.–An important medicinal plant | |
| Pandey et al. | In vitro propagation of Aconitum balfourii Stapf.: an important aconite of the Himalayan alpines | |
| CN103004595A (en) | Twig cuttage breeding method for ginseng fruit | |
| KR0134140B1 (en) | Garlic tissue cultivation | |
| CN106386507A (en) | Quick tissue-culture propagation method of dendrobium huoshanense seed seedlings | |
| Rajput et al. | Effective and large scale in vitro propagation of Dendrocalamus strictus (Roxb.) Nees using nodal segments as explants | |
| EP0380692A1 (en) | Plant tissue culture process | |
| CN108575746A (en) | A kind of Chinese herbaceous peony vitro Regeneration System method for building up | |
| Rao et al. | Propagation of bamboo and rattan through tissue culture | |
| KR100367104B1 (en) | Mass Production Methods of Somatic Embryos and Plantlets from Embryogenic Cells of Korean Ginseng by Suspension Culture | |
| Maulida et al. | Induction of kopyor coconut embryogenic callus using 2.4-D and TDZ | |
| CN105230493B (en) | A kind of propagation method of bighead atractylodes rhizome seedling and its application | |
| CN110810249B (en) | Culture medium for promoting elongation of oriental blueberry cluster buds as well as preparation method and application of culture medium | |
| CN101120654B (en) | Tissue culturing method for chia | |
| CN107711501A (en) | A kind of tissue culture and rapid propagation method of radix tylophorae | |
| Reghunath et al. | Micropropagation of cardamom (Elettaria cardamomum Maton) | |
| KR100533793B1 (en) | Mass propagation Method for Stock and Adventitious Root of Acanthopanax Koreanum Nakai Using Bioreactor | |
| KR101934780B1 (en) | Methods of cultivating plantlets in vitro derived from stem node of Moringa | |
| CN110896861A (en) | Tissue culture rapid propagation and seedling raising method for agastache rugosus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20071220 Year of fee payment: 6 |
|
| LAPS | Lapse due to unpaid annual fee |