CN1261563C - Method for freeze-drying and ultra low temperature storing ginseng callus cell - Google Patents
Method for freeze-drying and ultra low temperature storing ginseng callus cell Download PDFInfo
- Publication number
- CN1261563C CN1261563C CN03120982.3A CN03120982A CN1261563C CN 1261563 C CN1261563 C CN 1261563C CN 03120982 A CN03120982 A CN 03120982A CN 1261563 C CN1261563 C CN 1261563C
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- Prior art keywords
- cell
- protective material
- freeze
- ginseng callus
- drying
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 31
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 27
- 241000208340 Araliaceae Species 0.000 title claims abstract description 24
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 24
- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 24
- 235000008434 ginseng Nutrition 0.000 title claims abstract description 24
- 238000004108 freeze drying Methods 0.000 title claims abstract description 16
- 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 17
- 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 17
- 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 17
- 210000004027 cell Anatomy 0.000 claims description 52
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 39
- 239000000463 material Substances 0.000 claims description 36
- 230000001681 protective effect Effects 0.000 claims description 33
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 235000011187 glycerol Nutrition 0.000 claims description 11
- 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 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 8
- 238000002560 therapeutic procedure Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 210000004748 cultured cell Anatomy 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 2
- 239000003223 protective agent Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 9
- 230000012010 growth Effects 0.000 description 7
- 238000005201 scrubbing Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000005138 cryopreservation Methods 0.000 description 5
- 235000020183 skimmed milk Nutrition 0.000 description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 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 3
- 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 3
- 239000008103 glucose Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004992 fission Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022472 cold acclimation Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 235000013311 vegetables Nutrition 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
Landscapes
- 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)
- Dentistry (AREA)
- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a freeze drying and ultra-low temperature storage method of ginseng callus cells, which uses trehalose as a protective agent component for freeze drying and ultra-low temperature storage of ginseng cells. The present invention relates to preserved ginseng callus cells. With the method of the present invention, the effect for protecting the ginseng callus cells is obvious.
Description
Technical field:
The cell that the present invention relates to plant germplasm resource is preserved the particularly preservation of ginseng callus cell.The present invention relates to the freeze drying process and the cryopreservation method of ginseng callus cell, and freeze dried ginseng callus cell.
Background technology:
The plant genetic diversity is the protection environment for human survival and keeps a key factor of agricultural sustainable development, and how preserving and utilizing species diversity is one of key subjects of facing of current society.Plant tissue culture technique and very low temperature are preserved technology and are played an important role in the prolonged preservation of plant germplasm, and the some of them method has begun to enter application.So far also do not form a kind of both commonly used in the laboratory, the technique means that can preserve plant germplasm again on a large scale steadily in the long term simultaneously, it is further perfect to still need on some sport technique segments.
And for many vegetable cells, for example ginseng callus cell does not have any store method to be suggested as yet.
Summary of the invention
One of purpose of the present invention provides a kind of method of preservation plant germplasm, particularly ginseng callus cell.
Another object of the present invention provides a kind of recovery plant germplasm, as the method for ginseng callus cell.
Another object of the present invention provides a kind of plant germplasm of preservation, as ginseng callus cell.
Other purpose of the present invention is embodied among the detailed description of content of the present invention
According to the present invention, a kind of method of preserving ginseng callus cell comprises:
1) at low temperatures, ginseng callus cell is mixed with protective material,
2) mixture is handled with freeze-drying or ultralow warm therapy,
3) mixture after preservation is handled.
The protective material that adopts among the present invention contains trehalose, for example contains 10% trehalose; As required, can also add sucrose, for example add 10% sucrose.
According to one embodiment of the invention, this protective material contains 10% trehalose, 5-10% DMSO and 5-10% glycerine.
In the preferred embodiment of the present invention, this protective material contains 10% trehalose, 10% sucrose, 5-10%DMSO and 5-10% glycerine.
The method according to this invention, ginseng callus cell can be cultivated (substratum osmotic pressure, cold acclimation are gone down to posterity, improve in acceleration) to this cell in advance with before protective material mixes.This protective material and pre-cultured cell are by 1: 1-1: 3 volume ratio is mixed.
A kind of method of the cell growth that recovers to preserve comprises according to the present invention
1) at 40-60 ℃ of cell that thaws and preserve,
2) in substratum, cultivate the cell that thaws.
The invention still further relates to a kind of ginseng callus cell of preservation.
Fig. 1 is the schema of genseng callus cell of the present invention freeze-drying and cryopreservation method.
1. the present invention has adopted the freeze drying Techniques of preserving successfully to preserve ginseng callus, has Simple to operate, economical rationality, can preserve in enormous quantities the refrigeration material advantage.
2. the present invention has adopted the ultralow temperature Techniques of preserving successfully to preserve ginseng callus, has behaviour Do to recover the fast advantage of growth behind simple, the cell cryopreservation.
3. the present invention uses trehalose to preserve the protective agent component as plant cell, supports as plant germplasm The cell in source is preserved protective agent, particularly ginseng callus cell and is preserved protective agent, at present also not yet Report is arranged.
4. the invention provides desirable vitrifying protective agent, trehalose can with glycerine, DMSO, Be mixed with composite protectant in sucrose, glucose, bright skim milk, the proline and select each component Good combination makes the easier vitrifying state that enters of cell.
Embodiment
Further specify the present invention below in conjunction with embodiment.
Embodiment 1:
Treat the preparation of frozen material: use fluid suspension culture, callus cell is advisable to cultivate 5～7 days; Use solid culture, callus cell is advisable to cultivate 10～15 days.
Embodiment 2:
Protective material preparation: get trehalose, sucrose, glucose, bright skimmed milk, proline(Pro) and prepare the 1-6 protective material respectively, 1,2,4,5, No. 6 protective material uses the dissolving of 67V liquid nutrient medium, and No. 3 protective material directly uses bright skimmed milk dissolving.Carrying out freeze-drying and very low temperature respectively preserves.Each concentration is as follows:
No. 1 protective material: 5% DMSO, 10% glycerine
No. 2 protective materials: 20% sucrose, 5% DMSO, 10% glycerine
No. 3 protective materials: bright skimmed milk, 5% DMSO, 10% glycerine
No. 4 protective materials: 1.0M proline(Pro), 5% DMSO, 10% glycerine
No. 5 protective materials: 10% trehalose, 5% DMSO, 10% glycerine
No. 6 protective materials: 10% trehalose, 10% sucrose, 5% DMSO, 10% glycerine
Embodiment 3:
The pre-cultivation: get and treat that frozen material cultivates in advance in the substratum that adds trehalose, sucrose, glucose, bright skimmed milk, proline(Pro) composition; when filling a prescription freeze-stored cells as pressing 5, No. 6 protective materials; 25 ℃ of pre-3d that cultivate in the 67V substratum that adds 5%DMSO and 5% trehalose, 4 ℃ of pre-1d that cultivate in 5%DMSO and 10% trehalose 67V substratum again.
Embodiment 4:
The lyophilize store method: the volume mixture that (0 ℃) protective material and pre-cultured cell are pressed 1:3 on ice bath at first, its concentration is 1/4 of original content, slowly adds, and stirs gently, inhales behind the 10min and removes liquid.Then, ice bath is handled 5min in the protective material of original content, is that 1ml is sub-packed in the cillin bottle with every freeze pipe cell and protectant cumulative volume at last, and it is interior after pre-freeze to put Freeze Drying Equipment ,-28 ℃ of lyophilize 22h, and taking-up is put in-20 ℃ of refrigerators frozen.
Embodiment 5:
Cryopreservation method: at first (0 ℃) protective material and pre-cultured cell were by 1: 3 volume mixture on ice bath, and its concentration is 1/4 of original content, slowly added, and stirred gently, inhaled behind the 10min and removed liquid.Then, ice bath is handled 5min in the protective material of original content, and is last, is that lml is sub-packed in the frozen pipe of plastics with every freeze pipe cell and protectant cumulative volume, falls 1 ℃ with per minute, and extremely-50 ℃ are dropped in the liquid nitrogen frozen rapidly.
Embodiment 6:
Cell after frozen among the embodiment 4 is put into the liquid scrubbing substratum wash 10min, should dilute gradually during washing, the liquid scrubbing substratum is inhaled for the used 67V liquid nutrient medium of pre-cultivation and is removed washings, the liquid scrubbing substratum need place 0 ℃, and washing back cell changes over to and recovers on the used 67V solid medium of pre-cultivation to cultivate.Observation of cell recovers growing state.The cellular-restoring well-grown.
Embodiment 7
1min in 60 ℃ of water-baths thaws fast with the cell after frozen among the embodiment 5, cell mass is put into the liquid scrubbing substratum wash 10min, should dilute gradually during washing, the liquid scrubbing substratum is inhaled for the used 67V liquid nutrient medium of pre-cultivation and is removed washings, the liquid scrubbing substratum need place 0 ℃, and washing back cell changes over to and recovers on the used 67V solid medium of pre-cultivation to cultivate.Observation of cell recovers growing state.The cellular-restoring well-grown.
Presentation of results of the present invention:
Observe in the present invention when using 1,2,3,4,5, No. 6 protective material, adopt lyophilize and ultralow warm therapy depositary to join callus cell respectively, after through 0-6 month preservation period, thaw and cultivate again, the growth of part cellular-restoring is all arranged.But, use 5, No. 6 protective materials by freeze-drying and ultralow warm therapy preserve thaw after, the cellular-restoring phase is short, growth is very fast, just can be observed cell fission after week at 5-6 and grows fresh and tender new cell, and be better than for No. 6 No. 5.And use the 1-4 protective material by freeze-drying and ultralow warm therapy preserve thaw after, the cellular-restoring phase is longer, there is browning in various degree in cell, it is slower grow, at 8-10 after week, even for more time, just can observe cell fission and grow fresh and tender new cell.The present invention also observes and uses 5, No. 6 protective materials, preserves suspended culture cell by freeze-drying and ultralow warm therapy simultaneously, and ultralow warm therapy cellular-restoring growth is fast than lyophilization.
Description of test adopts the protective material that contains trehalose, be feasible with lyophilization and the frozen ginseng callus cell of ultralow warm therapy, and effect is fine.
When observation of cell recovers growth, because at present the most frequently used TTC method and FDA method detect cytoactive, the comprehensive action of enzyme in certain in the just cell that detects, and real cytoactive, also need keep cyto-architectural integrity, though very high through the relative survival rate of TTC method and the detection of FDA method, but be inoculated on the recovery media, may can not recover growth at all, illustrate that the endomembrane system of cell or other structures have suffered irreversible fatal damage.Therefore, adopt the most direct, the strongest, as to have actual value most method in the present invention, both the ratio of growth was recovered for judging the cell survival index in the frozen back of observation of cell.
Claims (6)
1, a kind of method of preserving ginseng callus cell comprises:
1) at low temperatures, ginseng callus cell is mixed with protective material,
2) mixture is handled with freeze-drying or ultralow warm therapy,
3) mixture after preservation is handled
Wherein this protective material contains trehalose.
2, the process of claim 1 wherein that this protective material contains 10% trehalose, 5-10%DMSO and 5-10% glycerine.
3, the method for claim 2, wherein this protective material also contains 10% sucrose.
4, according to the method for claim 3, wherein this protective material contains 10% trehalose, 10% sucrose, 5%DMSO and 10% glycerine.
5, according to the method one of among the claim 1-4, before further being included in ginseng callus cell and protective material mixing, this cell is cultivated in advance.
6, according to the method for claim 5, wherein this protective material and pre-cultured cell are by 1: 1-1: 3 volume ratio is mixed.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03120982.3A CN1261563C (en) | 2003-03-26 | 2003-03-26 | Method for freeze-drying and ultra low temperature storing ginseng callus cell |
| PCT/CN2004/000264 WO2004084629A1 (en) | 2003-03-26 | 2004-03-26 | The freeze-drying and crypreservation method of callus cell and the cryoprotectant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03120982.3A CN1261563C (en) | 2003-03-26 | 2003-03-26 | Method for freeze-drying and ultra low temperature storing ginseng callus cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1532279A CN1532279A (en) | 2004-09-29 |
| CN1261563C true CN1261563C (en) | 2006-06-28 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN03120982.3A Expired - Fee Related CN1261563C (en) | 2003-03-26 | 2003-03-26 | Method for freeze-drying and ultra low temperature storing ginseng callus cell |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1261563C (en) |
| WO (1) | WO2004084629A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424176C (en) * | 2005-06-03 | 2008-10-08 | 中国科学院上海生命科学研究院 | Ginseng tonolast aquaporin gene, its coded amino acid sequence and use |
| CN1962852B (en) * | 2006-11-27 | 2011-11-09 | 西北农林科技大学 | Use of gadol polysaccharide in preparation of antifreeze agent and its products and preparation method |
| CN101037666B (en) * | 2007-02-09 | 2010-04-21 | 浙江大学 | Low temperature preservation solution for animal cells and preserving method |
| CN102258008B (en) * | 2011-05-31 | 2013-08-21 | 成都大学 | Vitrification ultra-low temperature preserving method for effectively preserving bitter buckwheat callus |
| CN102258010B (en) * | 2011-09-02 | 2013-12-18 | 成都大学 | Slow refrigerating preservation method for paris polyphylla rootstock |
| CN102578077B (en) * | 2012-01-13 | 2013-07-24 | 成都美进生物科技有限公司 | Serum-free cryoprotectits agent |
| CN102763591B (en) * | 2012-07-05 | 2013-11-13 | 中国农业科学院作物科学研究所 | Atrac tylodes macrocephala koidz conservation method and re-cultivating method |
| CN103202225B (en) * | 2013-03-11 | 2014-07-30 | 上海市农业生物基因中心 | Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants |
| CN103283716B (en) * | 2013-06-08 | 2014-11-26 | 中南民族大学 | Simple cryopreservation method of plant BY-2 cell |
| CN105766894B (en) * | 2016-05-18 | 2018-10-19 | 中国农业科学院特产研究所 | Ginseng Multiple Buds cryopreservation and plant regeneration cultural method |
| CN115176708B (en) * | 2022-08-12 | 2023-07-04 | 江苏省中国科学院植物研究所 | Ultralow-temperature cryopreservation method for embryogenic callus of larch |
-
2003
- 2003-03-26 CN CN03120982.3A patent/CN1261563C/en not_active Expired - Fee Related
-
2004
- 2004-03-26 WO PCT/CN2004/000264 patent/WO2004084629A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| CN1532279A (en) | 2004-09-29 |
| WO2004084629A1 (en) | 2004-10-07 |
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Assignee: Changchun Keygen Biological Products Co., Ltd. Assignor: Changchun Research Institute of Biological Products Contract record no.: 2011220000024 Denomination of invention: Method for freeze-drying and ultra low temperature storing ginseng callus cell Granted publication date: 20060628 License type: Exclusive License Open date: 20040929 Record date: 20110801 |
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