WO2004084629A1 - Procede de lyophilisation et de cryoconservation de cals et cryoprotecteur utilise - Google Patents
Procede de lyophilisation et de cryoconservation de cals et cryoprotecteur utilise Download PDFInfo
- Publication number
- WO2004084629A1 WO2004084629A1 PCT/CN2004/000264 CN2004000264W WO2004084629A1 WO 2004084629 A1 WO2004084629 A1 WO 2004084629A1 CN 2004000264 W CN2004000264 W CN 2004000264W WO 2004084629 A1 WO2004084629 A1 WO 2004084629A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protective agent
- cells
- callus
- trehalose
- sucrose
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 54
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 49
- 238000004108 freeze drying Methods 0.000 title claims abstract description 15
- 239000002577 cryoprotective agent Substances 0.000 title abstract 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 24
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 24
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 24
- 238000004321 preservation Methods 0.000 claims abstract description 17
- 235000008434 ginseng Nutrition 0.000 claims abstract description 15
- 241000208340 Araliaceae Species 0.000 claims abstract description 14
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims abstract description 14
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 14
- 239000003223 protective agent Substances 0.000 claims description 51
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 37
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 34
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 27
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 22
- 229930006000 Sucrose Natural products 0.000 claims description 22
- 239000005720 sucrose Substances 0.000 claims description 22
- 230000012010 growth Effects 0.000 claims description 15
- 235000011187 glycerol Nutrition 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 10
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 9
- 241000196324 Embryophyta Species 0.000 claims description 9
- 239000000600 sorbitol Substances 0.000 claims description 9
- 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 claims description 7
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 7
- 229960000367 inositol Drugs 0.000 claims description 7
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 7
- 235000020183 skimmed milk Nutrition 0.000 claims description 7
- 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 claims description 6
- 238000012258 culturing Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 79
- 239000002609 medium Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 12
- 239000011814 protection agent Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 238000010257 thawing Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000007710 freezing Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000004017 vitrification Methods 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000004114 suspension culture Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 230000001012 protector Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000003934 vacuole Anatomy 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 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
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- KLDXJTOLSGUMSJ-JGWLITMVSA-N Isosorbide Chemical compound O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 KLDXJTOLSGUMSJ-JGWLITMVSA-N 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 240000004371 Panax ginseng Species 0.000 description 1
- 235000002789 Panax ginseng Nutrition 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 210000000633 nuclear envelope Anatomy 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
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- -1 sucrose Chemical compound 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Classifications
-
- 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
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- 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
Definitions
- the present invention relates to the preservation of cells of plant germplasm resources, especially callus cells.
- the present invention relates to a method for lyophilizing callus cells and a method for cryopreservation, a protective agent used in preservation, and preserved callus cells. Background technique
- Plant genetic diversity is a key factor in protecting the human living environment and maintaining sustainable agricultural development. How to preserve and use species diversity is one of the major issues facing society today. Plant tissue culture and preservation technologies, including freeze-drying and cryopreservation methods, play an important role in long-term preservation of plant germplasm, and some of these methods have begun to enter practical applications. However, a technical method that can be used in the laboratory and that can maintain plant germplasm on a large scale for a long period of time has not yet been formed, and some technical links need to be further improved.
- An object of the present invention is to provide a method for preserving callus cells.
- Another object of the present invention is to provide a protective agent for preserving plant callus cells.
- Another object of the present invention is to provide a method for recovering callus cells.
- Another object of the present invention is to provide a preserved callus cell.
- the invention provides a method for preserving callus cells, including:
- the cells are pre-cultured before optionally mixing the cells with the protective agent.
- the invention also provides callus cells preserved according to the method of the invention.
- the invention also provides a protective agent for preserving callus cells, the protective agent comprising trehalose.
- the protective agent of the present invention contains trehalose and one or more components selected from the group consisting of glucose, sucrose, skim milk, inositol, dimethylsulfoxide (DMSO), glycerol, and ethylene glycol.
- DMSO dimethylsulfoxide
- the invention also provides the use of a protective agent according to the invention in the preservation of callus cells.
- the present invention also provides a method for restoring the growth of preserved callus cells, including 1) thawing the preserved cells at about 36-45 ° C,
- the invention adopts freeze-drying preservation technology to successfully save callus cells, and has the advantages of simple operation, economical and reasonable, rapid growth recovery after frozen cells, and the ability to store frozen materials in large quantities.
- a method for storing callus cells including:
- the callus cells are mixed with the protective agent,
- the callus cells are preferably pre-cultured, at a low temperature, preferably at an ambient temperature to about 0 °. c, more preferably at about 5 ° C to about 0.
- the protective agent is mixed with the pre-cultured cells in a volume ratio of about 1: 1 to 1: 3.
- the mixture mixed with the protective agent is subjected to freeze-drying or ultra-low temperature processing, and then the obtained treated cells are respectively corresponding to about -20 ° C ⁇ 2 ° C or about -196 ° C.
- Liquid nitrogen liquid nitrogen device
- the preserved cells are rapidly thawed at a temperature of about 36-45 ° C, and recultured to restore the cells to growth.
- the preculture is a combination culture of callus cells in a sucrose preculture medium and a sorbitol preculture medium.
- a protective agent suitable for preserving callus cells for use in a freeze-drying method and a cryopreservation method.
- the protective agent contains trehalose in the culture medium and one or more components selected from the group consisting of glucose, sucrose, skim milk, inositol, DMSO, glycerol, and ethylene glycol.
- the protective agent used in the present invention contains trehalose, for example, contains 5-10% of trehalose, and sucrose may be added, for example, 10-20% of sucrose, if necessary.
- the protective agent contains 5-10% trehalose, 5-10% DMSO and 5-10% glycerol.
- the protective agent contains 5-10% trehalose, 10-20% sucrose, 5-10% DMSO, 5-10% glycerol, and 10-15% ethylene glycol. .
- protection is provided that contains 10% trehalose, 5% glucose, 10% skim milk, 5% v inositol, and optionally 10% sucrose Agent
- the protective agent is particularly suitable for lyophilization preservation; in another preferred embodiment of the present invention, provided with 10% trehalose 5% DMSO 10% glycerol, 10% ethylene glycol and optionally 10 % Sucrose protection agent, this protection agent is especially suitable for ultra-low temperature storage.
- the percentage content refers to the weight / volume percentage content, such as the percentage content in grams / ml.
- the protective agent of the present invention further contains one or more basal media.
- a preserved callus cell is also provided.
- a method for restoring preserved cell growth according to the present invention includes
- Example 1 The thawed cells are cultured in a medium.
- the present invention will be further described with reference to preferred embodiments. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the invention to the examples. Among them, the components used in the present invention are commercially available products.
- Example 1 the components used in the present invention are commercially available products.
- Disodium hydrogen phosphate Na 2 HP0 4 .123 ⁇ 40 50mg
- Pantothenic acid 4 mg l.OOmg
- Example 2 Sterilize at 121 ° C for 20 minutes. When using solid media, an additional 8 g of agar powder is required.
- Example 2
- Each component was dissolved in a 67 V liquid medium to prepare each protective agent.
- Example 3 Each component was dissolved in a 67V liquid medium to prepare a protective agent.
- Example 3 Each component was dissolved in a 67V liquid medium to prepare a protective agent.
- Pre-cultivation Take the ginseng callus to be frozen and store it in sucrose pre-culture medium (add 30 g / L sucrose in the passage medium) and sorbitol pre-culture medium (add 0.4 mol / L sorbitol in the passage medium) to cultivate. Suspension culture in sucrose pre-culture medium for 1 to 10 days, and suspension culture in sorbitol pre-culture medium for 1-3 days. Among them, 67V liquid was used as the passage medium Medium
- Example 4
- Freeze-drying preservation method First, in a water bath (0 ° C), mix the protective agent and the pre-cultured cells in a volume ratio of 1: 1 to 1-3, and keep it for 20-30 minutes. Finally, the total volume of cells and protective agent in each freezing tube was divided into 3ml aliquots in a 30ml vial, placed in a lyophilizer and pre-frozen at -28. C freeze-dry for 22h, remove and place at -20 ⁇ 2. C Frozen in water tank.
- Example 5 Example 5:
- Ultra-low temperature preservation method Put the pre-cultured suspension cells into a centrifuge tube, and after the cells sink, aspirate the culture medium, and then slowly add 25% of the original concentration protection agent on the ice bath, and gently stir, The ratio of the protective agent to the pre-cultured cells is 1: 1-3, and the liquid is aspirated after staying on the ice bath for 10-30 minutes. Then, still on the ice bath (0 ° C), slowly add 100% of the original concentration of protective agent, gently stir, and leave on the ice bath for 5-15min. After processing, the cells in the centrifuge tube were aliquoted into a freezer tube with a volume of 1ml, and the freezer tube was directly put into a liquid nitrogen freezing device.
- Example 6 Example 6:
- Example 4 The cells frozen in Example 4 were placed at room temperature, and each ampoule was washed with 67V liquid medium containing 1.5-2.0mol / L sorbitol for 3 times. 1: 3; 1: 5; 1: 10 (v / v), each wash time is 3-5min, should be gradually diluted during washing, after washing the cells are transferred to a 67V solid culture containing 1.0-1.5mol / L sorbitol Basal recovery culture. Observe the cells resume growth.
- Example 7 Example 7
- Example 5 The cells frozen in Example 5 were quickly thawed in a 36-45 ° C water bath for 1 minute, and then the protective agent in each freezing tube was removed by suction, and 1.5-2.0 mol / L sorbid was added to each freezing tube. Alcohol 67V liquid washing medium was washed 3 times, and the ratio of cells to washing solution was 1: 2; 1: 5; 1: 10 (v / v) in each washing, and the washing time was 3-5min each time. The cells were transferred to a 67 V solid medium containing 1.0-1.5 mol / L sorbitol to resume culture. Observe the cells resume growth. table 3 In the present invention, it was observed that when the lyophilized protection agent No.
- the ginseng callus was stored by freeze-drying method. After the 0-6 month storage period, after thawing and culturing, the No. 1, 2, and 3 were used. The cells did not resume growth after thawing the protective agent. The cells resumed growth after thawing with the protective agents No. 4 and 5, and the recovery period of No. 5 was short and the growth was fast. It was possible to observe that the cells divide into fresh and new cells in 6-7 weeks. No. 4 has a longer recovery period.
- the vitrification protector No. 6-11 when used, the ginseng callus cells were stored in an ultra-low temperature method, and after the 0-6 month storage period, after thawing and culturing, the 6-9 protector cells were used. None recovered. However, when the protection agents No. 10 and No. 11 were processed according to the ultra-low temperature method, it was found that they promoted the vitrification of the cells, and after thawing the preserved cells, the cell recovery period was short and the growth was faster, after 5-6 weeks You can observe that the cells divide and grow new and tender cells, and 11 is better than 10.
- the most commonly used TTC method and FDA method to detect cell activity only measure the combined effect of an enzyme in the cell, and the true cell activity also needs to maintain the integrity of the cell structure,
- the relative survival rate detected by the TTC method and the FDA method is high, but growth may not be restored at all when inoculated on the recovery medium, which indicates that the cell's inner membrane system or other structures have suffered irreversible fatal damage. Therefore, in the present invention, the most direct, most powerful, and most practical method is adopted, which is to observe the ratio of cells to resume growth after cryopreservation as an index for judging cell survival.
- the present invention uses trehalose as the main component of a plant cell protection agent, and is combined with a component selected from the group consisting of glucose, sucrose, skim milk, inositol, DMSO, glycerol, ethylene glycol, and sorbitol, which brings about Tissue cells, especially ginseng callus cells, have very unexpected preservation effects.
- the present invention provides an ideal vitrification protection agent. By combining one or more components of trehalose and glycerin, DMSO sucrose, glucose, skim milk, etc. into a compound protection agent, the cells can more easily enter the vitrification. State, promoted ultra-low temperature storage.
- the present invention by using a protective agent containing trehalose as a main component, the present invention successfully adopts freeze-drying and ultra-low temperature preservation technology to successfully save callus cells, has simple operation, is economical and reasonable, can store frozen materials in large quantities, and freezes cells. Restore the advantages of fast growth after storage.
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
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Description
愈伤组织细胞冻干及超低温保存方法和保护剂 技术领域
本发明涉及植物种质资源的细胞， 特别是愈伤组织细胞的保存。 本发明 涉及愈伤组织细胞的冻干方法和超低温保存方法、保存中用到的保护剂以及 经保存的愈伤组织细胞。 背景技术
植物遗传多样性是保护人类生存环境和维持农业持续发展的一个关键 因素， 如何保存和利用物种多样性是当今社会面临的重大课题之一。 植物组 织培养技术与保存技术， 包括冷冻干燥和超低温保存法， 在植物种质的长期 保存中有重要作用， 其中一些方法已开始进入实际应用。 但是至今还未形成 一种既能够在实验室使用， 同时又能够大规模长期稳定地保存植物种质的技 术手段， 在一些技术环节上尚需进一步完善。
而对于许多植物细胞，例如愈伤组织细胞，尚未有任何保存方法被提出。 本发明的发明人意外地发现使用海藻糖作为特别是愈伤组织细胞的植 物种质资源细胞保存保护剂组分 , 带来了意想不到的保存过程操作筒单、 可 大批量保存冷冻材料并且细胞冻存后恢复生长快效果。
发明概述
本发明的目的之一是提供一种保存愈伤组织细胞的方法。
本发明的又一目的是提供一种用于保存植愈伤組织细胞的保护剂。
本发明的另一目的是提供一种恢复愈伤组织细胞的方法。
本发明的另一目的是提供一种保存的愈伤组织细胞。
本发明提供了一种保存愈伤组织细胞的方法， 包括：
i)在低温下， 将愈伤组织细胞与保护剂混合，
ii)将混合物根据冷冻干燥法或超低温法进行处理，
iii)保存处理后的混合物，
其中该保护剂含有海藻糖， 任选地将细胞与保护剂混合前， 对该细胞进 行预培养。
本发明还提供了按本发明方法保存的愈伤组织细胞。
本发明还提供了一种用于保存愈伤组织细胞的保护剂，该保护剂包括海 藻糖。具体地，本发明的保护剂含有海藻糖和选自葡萄糖、蔗糖、脱脂牛奶、 肌醇、 二曱基亚砜 (DMSO)、 甘油和乙二醇中的一种或多种组分。
本发明还提供了根据本发明的保护剂在保存愈伤组织细胞中的用途。 另外， 本发明还提供了一种恢复保存的愈伤组织细胞生长的方法， 包括 1)在约 36-45°C解冻保存的细胞，
2)培养解冻的细胞。
本发明采用了冷冻干燥保存技术成功地保存了愈伤组织细胞,具有操作 简单、 经济合理、 细胞冻存后恢复生长快、 可大批量保存冷冻材料的优点。
本发明的其它目的 , 体现在本发明内容的详细描述之中。 发明详述
根据本发明， 提供了一种保存愈伤组织细胞的方法， 包括：
1)在低温, 优选在环境温度至 o。c的温度范围内， 将愈伤组织细胞与保 护剂混合，
2)将混合物根据冷冻干燥法或超低温法进行处理，
3)保存处理后的混合物。
根据本发明的一个优选技术方案中 , 在将愈伤组织细胞与保护剂进行混 合之前，优选将愈伤组织细胞进行预培养，在低温，优选在环境温度至约 o。c, 更优选在约 5°C至约 0。C的温度范围内， 加入保护剂， 进行混合。 优选， 保 护剂与预培养的细胞按约 1 : 1至 1 : 3的体积比进行混合。 接下来， 根据需 要， 将与保护剂混合后的混合物经冷冻干燥或者超低温法处理， 之后将所得 到的经处理的细胞相应地分别在约 -20°C±2°C或约 -196°C的液氮 (液氮器）中进 行保存。
根据本发明的恢复经保存的细胞生长的方法， 将经保存的细胞在约 36-45°C的温度下快速解冻， 进行再培养， 以使细胞恢复生长。
根据本发明的一个优选的实施方案，预培养是将愈伤组织细胞在蔗糖预 培养基和山梨醇预培养基中进行组合培养。
根据本发明，提供了适用于在冷冻干燥法和超低温保存法中使用的保存 愈伤组织细胞的保护剂。 该保护剂含有在培养基中的海藻糖和选自葡萄糖、 蔗糖、 脱脂牛奶、 肌醇、 DMSO、 甘油和乙二醇中的一种或多种组分。
根据本发明的一个实施方案， 本发明中采用的保护剂含有海藻糖， 例如 含有 5-10%的海藻糖， 根据需要， 还可以添加蔗糖， 例如添加 10-20%的蔗 糖。 根据本发明的另一个实施方案， 该保护剂含有 5-10%的海藻糖， 5-10% 的 DMSO和 5-10%的甘油。 根据本发明的一个优选实施方案， 该保护剂含 有 5-10%的海藻糖， 10-20%的蔗糖，5-10% 的 DMSO、5-10%的甘油和 10-15% 的乙二醇。 特别地， 在本发明的一个优选实施方案中， 提供了含有 10%的海 藻糖、 5%的葡萄糖、 10% 的脱脂牛奶、 5%v的肌醇， 以及任选的 10 %的蔗 糖的保护剂， 该保护剂特別适用于冻干保存; 在本发明另一个优选实施方案 中， 提供了含有 10%的海藻糖 5% 的 DMSO 10%的甘油、 10%的乙二醇 以及任选的 10 %的蔗糖的保护剂，该保护剂特别适用于超低温保存。在本发 明中， 除非特别指明， 所述百分含量指的是重量 /体积百分含量， 如克 /毫升 的百分含量。
在本发明的保护剂中， 还含有一种或多种基础培养基。
根据本发明， 还提供了一种保存的愈伤组织细胞。
根据本发明的恢复保存的细胞生长的方法， 包括
1)在约 36-45°C解冻保存的细胞，
2)在培养基中培养解冻的细胞。 下面结合优选的实施例进一步说明本发明。 但是应该明白， 这些实施例 仅是用于说明的目的， 并不是打算将本发明限制到实施例。 其中， 本发明中 所使用的组分为可市售购得的产品。 实施例 1 :
待冻存材料的准备：将人参 (panax ginseng CA. Meyer)愈伤组织细胞用液 体培养基进行悬浮培养， 培养 5〜7 天为宜； 用固体培养基进行培养， 培养
天为宜。 此处所使用的液体培养基为 67V液体培养基， 每 L含有: 组分 含量 (mg)
磷酸二氢钠 (Na¾P04.2H20) 195mg
磷酸氢二钠 (Na2HP04.12¾0) 50mg
氯化钾 (KC1) 200mg
硫酸鎂 (MgS04.7H20) 250mg
硫酸铵 ((NH4)2S04) lOOmg
硝酸钾 (KN03) 800mg
氯化钙 (CaCl2'2H20) 200mg
钼酸钠 (Na2Mo04'2H20) 0.25mg
硫酸铜 (CuS04'5H20) 0.25mg
硫酸锰 (MnS04'H20) 4.00mg
硫酸锌 (ZnS04'7¾0) 5.95mg
氯化钴 (CoCl2'6H20) 4.83mg
硼酸 (H3B03) 5.00mg
碘化钾 (KI) 0.05mg
盐酸疏胺素 0.5mg
盐酸吡哆醇 0.5mg
烟酸 1.25mg
泛酸 4丐 l.OOmg
硫酸亚铁 (FeS04'7H20) 13.9mg
乙二胺四乙酸二钠 (Na2EDTA) 18.5mg
肌醇 lOOmg
蔗糖 30g
乳清蛋白 ig
H 约 5.8
121 °C高温湿热灭菌 20min。 当使用固体培养基时， 还需要再加入 8g琼脂粉。
实施例 2:
保护剂配制：
表 1: 冻干保护剂的配制 (冷冻千燥法)
将各组分使用 67V液体培养基溶解而制备各保护剂。
将各组分使用 67V液体培养基溶解而制备保护剂。 实施例 3:
预培养： 取待冻存人参愈伤组织在蔗糖预培养基 (传代培养基中 再加 30g/L蔗糖）和山梨醇预培养基（传代培养基中再加 0.4mol/L山 梨醇）中组合培养。 在蔗糖预培养基中悬浮培养 1- 10天， 在山梨醇 预培养基中悬浮培养 1-3天。 其中， 传代培养基使用的是 67V液体
培养基 实施例 4:
冷冻干燥保存方法： 首先在水浴上 (0。C), 将保护剂与预培养细胞按 1 : 1-3的体积比进行混合， 保持 20-30min。 最后以每冷冻管细胞和保护剂的总 体积为 3ml分装于 30ml西林瓶中，置冻干机内经预冻后，于 -28。C冷冻干燥 22h, 取出置于 -20±2。C水箱中冻存。 实施例 5:
超低温保存方法: 将预培养悬浮细胞放入离心管中， 待细胞下沉后吸去 培养基， 然后在冰浴上 (0。C)， 緩緩加入 25%原浓度保护剂, 轻轻搅拌， 保 护剂与预培养细胞的比例为 1 : 1-3 , 冰浴上停留 10-30min后吸去液体。 然 后， 仍在冰浴上 (0°C), 緩緩加入 100%原浓度保护剂 , 轻轻搅拌, 冰浴上停 留 5-15min。处理完毕后将离心管中的细胞分装于冷冻管中，分装体积为 lml， 将冷冻管直接投入液氮器中冻存。 实施例 6:
将实施例 4中冻存后的细胞放至室温，在每支安瓿中加入含 1.5-2.0mol/L 山梨醇的 67V液体培养基洗涤 3次，每次洗涤时细胞与洗涤液的比例分别为 1 : 3; 1 : 5; 1 : 10 ( v/v ), 每次洗涤时间为 3-5min, 洗涤时应逐渐稀释， 洗涤后细胞转入含 1.0-1.5mol/L山梨醇的 67V固体培养基上恢复培养。观察 细胞恢复生长情况。 实施例 7
将实施例 5中冻存后的细胞在 36-45°C水浴中 lmin内快速化冻, 然后 吸去每支冷冻管中的保护剂， 在每个冷冻管中加入含 1.5-2.0mol/L 山梨醇 67V液体洗涤培养基洗涤 3次，每次洗涤时细胞与洗涤液的比例分别为 1 : 2; 1 : 5; 1 : 10 ( v/v ),每次洗涤时间为 3-5min, 洗涤后细胞转入含 1.0-1.5mol/L 山梨醇的 67V固体培养基上恢复培养。 观察细胞恢复生长情况。
表 3
在本发明中观察到当使用 1-5号冻干保护剂 , 采用冷冻干燥法保存人参 愈伤组织， 在经过 0-6月保存期后， 解冻再培养后， 其中使用 1、 2、 3号保 护剂解冻后细胞均不恢复生长， 使用 4、 5号保护剂解冻后细胞恢复生长， 而且 5号恢复期短， 生长较快， 6-7周便可观察到细胞分裂出鲜嫩的新细胞， 4号恢复期较长。
在本发明中观察到当使用 6-11号玻璃化保护剂，超低温法保存人参愈伤 组织细胞， 在经过 0-6月保存期后， 解冻再培养后， 其中使用 6-9号保护剂 细胞均不恢复生长。 但是， 使用 10、 11 号保护剂按超低温法进行处理时， 发现其促进了细胞形成玻璃化状态， 并且在对保存细胞进行解冻后， 细胞恢 复期短， 生长较快， 在 5-6周后便可观察到细胞分裂长出鲜嫩的新细胞， 而 且 11号更优于 10号。
本实验还通过透视电镜观察了化冻细胞在恢复生长 3天后细胞结构， 其 结构基本与未冻对照相似。 线粒体的膨大和基质稀少已被修复， 核膜清晰可 见， 液泡也重新变得大小不一， 小液泡增多。
在观察细胞恢复生长时， 由于目前最常用的 TTC法和 FDA法检测细胞 活性， 检测的只是细胞内某种酶的综合作用， 而真正的细胞活性， 还需保持 细胞结构的完整性， 即使经 TTC法和 FDA法检测的相对存活率很高， 但接 种到恢复培养基上， 也可能根本不能恢复生长， 这说明细胞的内膜系统或其 他结构已遭受不可逆的致命损伤。 因此， 在本发明中采用最直接、 最有力、 最具有实际价值的方法， 既观察细胞冻存后恢复生长的比率为判定细胞存活 指标。
实验说明， 采用含海藻糖与蔗糖等其它成分复合的保护剂， 用冻干法和 超低温法冻存人参愈伤组织细胞是可行的， 并且效果很好。
根据上述内容， 很显然：
本发明使用海藻糖作为植物细胞保存保护剂主要组分， 与选自包括葡萄 糖、 蔗糖、 脱脂牛奶、 肌醇、 DMSO、 甘油、 乙二醇、 山梨醇的组分组合， 带来了对于愈伤组织细胞，特别是人参愈伤组织细胞而言非常意想不到的保 存效果。 并且本发明提供了理想的玻璃化保护剂， 通过将海藻糖和甘油、 DMSO 蔗糖、 葡萄糖、 脱脂牛奶等中的一种或多种组分组合配制成复合保 护剂， 使细胞更容易进入玻璃化状态， 促进了超低温保存。 所以， 本发明通 过使用含有海藻糖作为主要成分的保护剂本发明采用了冷冻干燥和超低温 保存技术成功地保存了愈伤组织细胞, 具有操作简单、 经济合理、 可大批量 保存冷冻材料且细胞冻存后恢复生长快的优点。
Claims
1、 一种保存愈伤组织细胞的方法， 包括：
1)在低温下， 将植物愈伤组织细胞与保护剂混合，
2)将混合物根据冷冻干燥法或超低温法进行处理，
3)保存处理后的混合物，
其中该保护剂含有海藻糖。
2、 权利要求 1 的方法， 其中该保护剂含有海藻糖和选自包括下列的组 中的一种或多种组分： 葡萄糖、 蔗糖、 脱脂牛奶、 肌醇、 DMSO、 甘油和乙 二醇。
3、 权利要求 2的方法， 其中该保护剂含有约 5-10%海藻糖， 约 5-10% DMSO、 约 5-10%甘油和约 10-15%乙二醇。
4、 权利要求 3的方法， 其中该保护剂还含有约 10-20%蔗糖。
5、 权利要求 2的方法， 其中该保护剂含有约 5-10%海藻糖、 约 10-20% 蔗糖、 约 5- 10% DMSO和约 5- 10%甘油。
6、 权利要求 1-5 中之一的方法， 进一步包括在愈伤组织细胞与保护剂 混合前， 对该细胞进行预培养。
7、 权利要求 1-6 中之一的方法， 其中预培养是将细胞在蔗糖预培养基 和山梨醇预培养基中培养。
8、 权利要求 1-7中之一的方法， 其中该保护剂与预培养后细胞按约 1 :
1-1 : 3的体积比混合。
9、 权利要求 1-8中之一的方法，其中所述愈伤组织细胞是人参愈伤組织 细胞。
10、 一种按权利要求 1-9中之一的方法保存的愈伤组织细胞, 特别是人 参愈伤组织细胞。
11、 一种用于保存愈伤组织细胞， 特别是人参愈伤组织细胞的保护剂， 所述保护剂包括海藻糖。
12、 权利要求 11 的保护剂， 其中该保护剂含有海藻糖和选自包括下列 组中的一种或多种组分： 葡萄糖、 蔗糖、 脱脂牛奶、 肌醇、 DMSO、 甘油和 乙二醇。
13、权利要求 12的方法，其中该保护剂含有约 5-10%海藻糖， 约 5-10% DMSO、 约 5-10%甘油和约 10-15%乙二醇。
14、 权利要求 13的方法， 其中该保护剂还含有约 10-20%蔗糖。
15、权利要求 12的方法，其中该保护剂含有约 5-10%海藻糖、约 10-20% 蔗糖、 约 5-10% DMSO和约 5-10%甘油。
16、 权利要求 12-15中之一的保护剂在保存， 特别是冷冻干燥保存和超 低温保存愈伤组织细胞， 特别是人参愈伤组织细胞中的用途。
17、 一种恢复保存的愈伤组织细胞， 特别是人参愈伤组织细胞生长的方 法， 包括
1)在约 36-45°C解冻保存的细胞，
2)培养解冻的细胞。
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| CN102763591A (zh) * | 2012-07-05 | 2012-11-07 | 中国农业科学院作物科学研究所 | 一种白术的保存方法以及再培养方法 |
| CN103202225A (zh) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | 一种植物微茎尖超低温保存和解冻、恢复培养的方法 |
| CN115176708A (zh) * | 2022-08-12 | 2022-10-14 | 江苏省中国科学院植物研究所 | 一种落羽杉胚性愈伤组织的超低温冷冻保存方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424176C (zh) * | 2005-06-03 | 2008-10-08 | 中国科学院上海生命科学研究院 | 人参液泡膜水孔蛋白基因、其编码的氨基酸序列和应用 |
| CN1962852B (zh) * | 2006-11-27 | 2011-11-09 | 西北农林科技大学 | 红景天多糖在制备抗冻剂中的新用途及其产品和制备方法 |
| CN101037666B (zh) * | 2007-02-09 | 2010-04-21 | 浙江大学 | 一种动物细胞的低温保存溶液及保存方法 |
| CN102258008B (zh) * | 2011-05-31 | 2013-08-21 | 成都大学 | 一种从有效保存苦荞麦愈伤组织的玻璃化超低温保存方法 |
| CN102258010B (zh) * | 2011-09-02 | 2013-12-18 | 成都大学 | 一种华重楼根茎的慢速冷冻保存方法 |
| CN102578077B (zh) * | 2012-01-13 | 2013-07-24 | 成都美进生物科技有限公司 | 一种无血清低温保护剂 |
| CN103283716B (zh) * | 2013-06-08 | 2014-11-26 | 中南民族大学 | 一种简便的植物by-2细胞冷冻保存方法 |
| CN105766894B (zh) * | 2016-05-18 | 2018-10-19 | 中国农业科学院特产研究所 | 人参丛生芽超低温保存及植株再生培养方法 |
-
2003
- 2003-03-26 CN CN03120982.3A patent/CN1261563C/zh not_active Expired - Fee Related
-
2004
- 2004-03-26 WO PCT/CN2004/000264 patent/WO2004084629A1/zh active Application Filing
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102763591A (zh) * | 2012-07-05 | 2012-11-07 | 中国农业科学院作物科学研究所 | 一种白术的保存方法以及再培养方法 |
| CN102763591B (zh) * | 2012-07-05 | 2013-11-13 | 中国农业科学院作物科学研究所 | 一种白术的保存方法以及再培养方法 |
| CN103202225A (zh) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | 一种植物微茎尖超低温保存和解冻、恢复培养的方法 |
| CN115176708A (zh) * | 2022-08-12 | 2022-10-14 | 江苏省中国科学院植物研究所 | 一种落羽杉胚性愈伤组织的超低温冷冻保存方法 |
| CN115176708B (zh) * | 2022-08-12 | 2023-07-04 | 江苏省中国科学院植物研究所 | 一种落羽杉胚性愈伤组织的超低温冷冻保存方法 |
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| Publication number | Publication date |
|---|---|
| CN1532279A (zh) | 2004-09-29 |
| CN1261563C (zh) | 2006-06-28 |
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