CN107853293A - A kind of radix ampelopsis seed cryopreservation method - Google Patents
A kind of radix ampelopsis seed cryopreservation method Download PDFInfo
- Publication number
- CN107853293A CN107853293A CN201711297635.2A CN201711297635A CN107853293A CN 107853293 A CN107853293 A CN 107853293A CN 201711297635 A CN201711297635 A CN 201711297635A CN 107853293 A CN107853293 A CN 107853293A
- Authority
- CN
- China
- Prior art keywords
- radix ampelopsis
- seed
- cooling box
- liquid nitrogen
- room temperature
- Prior art date
- 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.)
- Pending
Links
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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
For the deficiency of existing radix ampelopsis Germ-plasma resources protection technology, the present invention provides a kind of ultra-low temperature preservation method for being suitable for radix ampelopsis seed, can be with the preservation radix ampelopsis germ plasm resource of long-term safety：Radix ampelopsis seed is sterilized, fluid nutrient medium soaks, is put into agent treatment in cryopreservation tube, is put into gradient cooling box, low temperature refrigerator cools, is put into liquid nitrogen, carries out liquid nitrogen cryogenics and saves backup.With technique is simple, stability is reliable, preserve after percentage of seedgermination and the germination percentage of fresh kind there was no significant difference, radix ampelopsis percentage of seedgermination is up to more than 95% after germination test result shows cryopreservation.
Description
Technical field
The invention belongs to modern Chinese herbal medicine cultivation technology field, and in particular to a kind of radix ampelopsis seed cryopreservation method.
Background technology
Radix ampelopsis is called five pawl rattans, goose hatches, cat ovum, porcupine waist, belong to Vitaceae Ampelopsis fall leaves for many years liana plant
Thing, hillside sylvan life is grown on, is distributed on the northeast in China, North China, south China and other places.Radix ampelopsis is traditional Chinese medicine, herb and
Bulk, which expands root, to be used as medicine, and its is sweet, cold nature, there is the effect of clearing heat and detoxicating, myogenic relieves the pain, subsided a swelling, and be used for curing carbuncle swells
Sore, scald, bruise, cellulitis, lymphnoditis etc..Modern medicine study proves that radix ampelopsis also has tender white muscle skin
Effect, the additive as cosmetics and skincare product use.In addition to medical value, the leaf peculiar beauty of radix ampelopsis, the elegant person of outstanding talent of plant shape
Show, it can be utilized for the vertical planes such as wall, frame, loggia, hedge wall, arched door, lamppost, naked rock, artificial hillock, trunk, roof, balcony
Greening, there is ornamental well and sight.If its sturdy root tuber loose, that form is totally different is transplanted among basin alms bowl, just
Can turn into-basin Gen Qiyemei, effect it is unique view and admire potted landscape.Also agricultural chemicals can be used as；Rhizome is starch-containing, can make wine.Due to radix ampelopsis
With important economic value, people are excavated without restraint to it in recent years, the abundance of wild radix ampelopsis is reduced rapidly, now
It is very serious to the destruction of radix ampelopsis germ plasm resource it is difficult to excavate wild radix ampelopsis, at present to being ground in terms of Chinese medicine germ plasm resource
Study carefully less.
Germ-plasma resources protection mode is normal temperature, Cord blood radix ampelopsis seed two ways at present.Normal temperature preserves Chinese medicine assortment
Son, a period of time is often preserved with regard to needing Regeneration again, because Chinese medicine seed reduces through preservation after a while, vigor
Quickly；Cord blood Chinese medicine seed, in China, national middle term storehouse and National crop genebank of China are established, national middle term storehouse is protected
The temperature deposited is 4 DEG C or so, and relative humidity is less than 65%, and the life-span for being typically designed stored seedses is 5~10 years, national genebank
Preserve storehouse for a long time for germplasm, the temperature in storehouse is -18 DEG C or so, and relative humidity is less than 50%, design shelf-life up to 20 years with
On, that is to say, that germplasm preserves in long-term storehouse, and vigor reduces likewise as the passage of time, and germplasm generally requires more after 20 years
New breeding, it is not permanent preservation.
Therefore, for securely held radix ampelopsis germ plasm resource, research and probe is a kind of safe and preserves radix ampelopsis germ plasm resource for a long time
Method highly significant, it can reduce or avoid seed update breeding, it is time saving and energy saving, block Regeneration during plant
Genetic drift etc. produces blastation between matter, ensures the genetic stability of germplasm.
The content of the invention
It is an object of the invention to provide a kind of ultra-low temperature preservation method for being suitable for radix ampelopsis seed, this method can supplement now
The deficiency of radix ampelopsis Germ-plasma resources protection technology, can be radix ampelopsis Germ-plasma resources protection with the preservation radix ampelopsis germ plasm resource of long-term safety
One new effective technological approaches is provided.
It is an object of the invention to provide a kind of radix ampelopsis seed cryopreservation method, including below step：
(1) radix ampelopsis seed is carried out disinfection with 70% alcohol at room temperature, carried out with 1~4% carboxymethylcellulose sodium solution
10~30min of seed soaking, after soaking 20~60min with white fluid nutrient mediums, add final concentration of 1.5~2.5mol/
L glycerol and 1~1.5mol/L dimethyl sulfoxide (DMSO)s, after continuing with 20~60min, take out treated radix ampelopsis seed；
(2) treated radix ampelopsis seed is put into cryopreservation tube, add containing 2.6~3.8mol/L glycerine, 1.5~
The white liquid mediums of 2mol/L dimethyl sulfoxide (DMSO)s and 0.45~0.6mol/L sucrose, 10~30min of incubation at room temperature, then will
It is put into gradient cooling box；
(3) the gradient cooling box with radix ampelopsis seed is put into low temperature refrigerator, makes the temperature of its cooling box at -35 DEG C
~-65 DEG C, cooling rate is -1 DEG C/min, then takes out cooling box, this cooling box is put into liquid nitrogen at once, and it is low to carry out liquid nitrogen
Temperature saves backup.
In a concrete scheme, contain 100% isopropanol or glycerine described in step (2) in gradient cooling box, can be with
Reuse.
In a concrete scheme, the temperature requirement of low temperature refrigerator is typically below -70 DEG C described in step (3).
When above-mentioned preserved radix ampelopsis seed is thawed, if room temperature is taken out more than 20 DEG C, by cryopreservation tube from liquid nitrogen, directly
Connect and utilize room temperature naturally to thaw；If room temperature is less than 20 DEG C, cryopreservation tube is taken out from liquid nitrogen, is placed in 20~24 DEG C of water baths
Thaw.After having thawed, radix ampelopsis seed in cryopreservation tube is taken out, then with running water or distills water washing 3~5 times, radix ampelopsis after cleaning
Seed is directly used in culture.
Technique effect
1st, cryopreservation radix ampelopsis seed method provided by the invention have technique is simple, stability is reliable, preserve after plant
There was no significant difference for sub- germination percentage and the germination percentage of fresh kind, and germination test result shows radix ampelopsis percentage of seedgermination after cryopreservation
Up to more than 95%.
2nd, the present invention has long-term, effective, securely held radix ampelopsis seed, and it is most effective to be that radix ampelopsis germ plasm resource preserves for a long time
Method.
Embodiment
Below, the present invention will be further detailed with embodiment, but it is not limited to any of these embodiments
One or similar example.
Embodiment 1
The radix ampelopsis harvested then is taken, is dried naturally, the radix ampelopsis seed of water content 12% or so is disappeared with 70% alcohol
Poison, seed soaking 20min is carried out with 1.5% carboxymethylcellulose sodium solution, with containing 2.1mol/L glycerine and 1.25mol/L bis-
Methyl sulfoxide white liquid mediums processing, handle 20min after, take out treated radix ampelopsis seed, place into containing
In the cryopreservation tube of the white liquid mediums of 3.15mol/L glycerine, 1.75mol/L dimethyl sulfoxide (DMSO)s and 0.5mol/L sucrose, room
Temperature incubates 30min, then puts it into gradient cooling box；Radix ampelopsis puts gradient cooling box into -70 DEG C of low temperature refrigerators and carries out gradient
Cooling, making the temperature of its cooling box, cooling rate is -1 DEG C/min, then takes out cooling box, at once by this cooling box at -50 DEG C
It is put into liquid nitrogen, after carrying out liquid nitrogen cryogenics preservation 5 years, cryopreservation tube is taken out from liquid nitrogen, places room temperature at 23 DEG C or so, directly
Using room temperature naturally to thaw, after having thawed, radix ampelopsis seed in cryopreservation tube is taken out, then with running water or distills water washing 3 times, clearly
Wash rear radix ampelopsis seed and be directly used in Seed germination, germination percentage reaches 96.58% after tested.
Comparative examples 1
The radix ampelopsis harvested then is taken, is dried naturally, the radix ampelopsis seed of water content 12% or so is disappeared with 70% alcohol
Poison, seed soaking 20min is carried out with 1.5% carboxymethylcellulose sodium solution, trained with the white liquid containing 2.1mol/L glycerine
Nutrient solution processing, after handling 20min, takes out treated radix ampelopsis seed, places into containing 3.15mol/L glycerine, 1.75mol/L bis-
In the cryopreservation tube of the white liquid mediums of methyl sulfoxide and 0.5mol/L sucrose, then put it into gradient cooling box；By ladder
Degree cooling box puts -70 DEG C of low temperature refrigerators into and carries out gradient coolings, makes the temperature of its cooling box at -50 DEG C, and cooling rate is -1 DEG C/
Min, then take out cooling box, this cooling box be put into liquid nitrogen at once, carry out liquid nitrogen cryogenics preserve 5 years after, by cryopreservation tube from
Taken out in liquid nitrogen, place room temperature at 23 DEG C or so, directly utilize room temperature naturally to thaw, after having thawed, take out radix ampelopsis in cryopreservation tube
Seed, then with running water or distill water washing 3 times, radix ampelopsis seed is directly used in Seed germination after cleaning, after tested germination percentage
Reach 79.51%.
Comparative examples 2
The radix ampelopsis harvested then is taken, is dried naturally, the radix ampelopsis seed of water content 12% or so is disappeared with 70% alcohol
Poison, seed soaking 20min is carried out with 1.5% carboxymethylcellulose sodium solution, with containing 2.1mol/L glycerine and 1.25mol/L bis-
Methyl sulfoxide white liquid mediums processing, handle 20min after, take out treated radix ampelopsis seed, place into containing
In the cryopreservation tube of the white liquid mediums of 3.15mol/L glycerine, 1.75mol/L dimethyl sulfoxide (DMSO)s and 0.5mol/L sucrose, room
Temperature incubates 30min, then puts it into after -5 DEG C or so low temperature refrigerators preserve 5 years, cryopreservation tube is taken out from liquid nitrogen, holding chamber
Temperature directly utilizes room temperature naturally to thaw at 23 DEG C or so, after having thawed, radix ampelopsis seed in taking-up cryopreservation tube, then with running water or
Person distills water washing 3 times, and radix ampelopsis seed is directly used in Seed germination after cleaning, and germination percentage reaches 51.25% after tested.And take new
Cord blood is after 5 years at fresh 1~5 DEG C of radix ampelopsis, and for Seed germination, radix ampelopsis fails to germinate after tested.
Embodiment 2
The radix ampelopsis harvested then is taken, is dried naturally, the radix ampelopsis seed of water content 8.5% or so is disappeared with 70% alcohol
Poison, seed soaking 15min is carried out with 2.5% carboxymethylcellulose sodium solution, with containing 2mol/L glycerine and 1.6mol/L diformazans
Base sulfoxide white liquid mediums processing, handle 30min after, take out treated radix ampelopsis seed, place into containing
In the cryopreservation tube of the white liquid mediums of 3.1mol/L glycerine, 1.7mol/L dimethyl sulfoxide (DMSO)s and 0.6mol/L sucrose, room temperature
20min is incubated, then is put it into gradient cooling box；Put gradient cooling box into -80 DEG C of low temperature refrigerators and carry out gradient cooling, make
For the temperature of its cooling box at -55 DEG C or so, cooling rate is -1 DEG C/min, then takes out cooling box, at once puts this cooling box
Enter in liquid nitrogen, after carrying out liquid nitrogen cryogenics preservation 6 years, cryopreservation tube is taken out from liquid nitrogen, place room temperature at 23 DEG C or so, directly profit
With room temperature naturally to thaw, after having thawed, radix ampelopsis seed in cryopreservation tube is taken out, then with running water or distills water washing 4 times, cleaning
Radix ampelopsis seed is directly used in Seed germination afterwards, and germination percentage reaches 98.01% after tested.
Claims (5)
1. it is an object of the invention to provide a kind of radix ampelopsis seed cryopreservation method, including below step：
(1) radix ampelopsis seed is carried out disinfection with 70% alcohol at room temperature, after soaking 20~60min with white fluid nutrient mediums, then
Final concentration of 1.5~2.5mol/L glycerine and 1~1.5mol/L dimethyl sulfoxide (DMSO)s are added, after continuing with 20~60min, is taken out
Treated radix ampelopsis seed；
(2) treated radix ampelopsis seed is put into cryopreservation tube, is added containing 2.6~3.8mol/L glycerine, 1.5~2mol/L bis-
The white liquid mediums of methyl sulfoxide and 0.45~0.6mol/L sucrose, 10~30min of incubation at room temperature, then put it into ladder
Spend in cooling box；
(3) the gradient cooling box with radix ampelopsis seed is put into low temperature refrigerator, makes the temperature of its cooling box -35 DEG C~-65
DEG C, cooling rate is -1 DEG C/min, then takes out cooling box, this cooling box is put into liquid nitrogen at once, carries out liquid nitrogen cryogenics guarantor
Deposit standby.
2. method according to claim 1, wherein the radix ampelopsis seed dries, seed moisture content naturally to harvest fresh kind then
For 6~15%.
3. contain 100% isopropanol or glycerine described in method according to claim 1, wherein step (2) in gradient cooling box,
It may be reused.
4. the temperature requirement of low temperature refrigerator is typically below -70 DEG C described in method according to claim 1, wherein step (3).
5. method according to claim 1, wherein when the radix ampelopsis seed preserved is thawed, if room temperature will freeze more than 20 DEG C
Deposit pipe to take out from liquid nitrogen, directly utilize room temperature naturally to thaw；If room temperature is less than 20 DEG C, cryopreservation tube is taken out from liquid nitrogen, and
It is placed in 20~24 DEG C of water baths and thaws.After having thawed, radix ampelopsis seed in cryopreservation tube is taken out, then washed with running water or distillation
Wash 3~5 times, radix ampelopsis seed is directly used in culture after cleaning.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711297635.2A CN107853293A (en) | 2017-12-08 | 2017-12-08 | A kind of radix ampelopsis seed cryopreservation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711297635.2A CN107853293A (en) | 2017-12-08 | 2017-12-08 | A kind of radix ampelopsis seed cryopreservation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107853293A true CN107853293A (en) | 2018-03-30 |
Family
ID=61705650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711297635.2A Pending CN107853293A (en) | 2017-12-08 | 2017-12-08 | A kind of radix ampelopsis seed cryopreservation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107853293A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103975852A (en) * | 2014-04-16 | 2014-08-13 | 西北大学 | Method for ultra low temperature storage of Saussurea involucrata Kar.et Kir callus |
| CN104222072A (en) * | 2014-08-27 | 2014-12-24 | 中国农业科学院麻类研究所 | Vitrification ultralow-temperature preservation method of flax seeds |
-
2017
- 2017-12-08 CN CN201711297635.2A patent/CN107853293A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103975852A (en) * | 2014-04-16 | 2014-08-13 | 西北大学 | Method for ultra low temperature storage of Saussurea involucrata Kar.et Kir callus |
| CN104222072A (en) * | 2014-08-27 | 2014-12-24 | 中国农业科学院麻类研究所 | Vitrification ultralow-temperature preservation method of flax seeds |
Non-Patent Citations (4)
| Title |
|---|
| D.H.TOUCHELL 等: "Cryopreservation of seed of Western Australian native species", 《BIODIVERSITY AND CONSERVATION 2》 * |
| 于丽杰 等: "《植物组织培养教程》", 31 August 2015, 华中科技大学出版社 * |
| 李春龙 等: "《植物组织培养》", 31 July 2016, 西南交通大学出版社 * |
| 黄璐琦 等: "《药用植物种质资源研究》", 31 December 2008, 上海科学技术出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101401568B (en) | Cryopreservation method for chrysanthemum | |
| Mutawila et al. | Optimisation of time of application of Trichoderma biocontrol agents for protection of grapevine pruning wounds | |
| CN102511351B (en) | Preventive method for kiwifruit canker | |
| CN103975852A (en) | Method for ultra low temperature storage of Saussurea involucrata Kar.et Kir callus | |
| CN101530053A (en) | Method for transplanting wild big myrica esculenta | |
| CN102726189A (en) | Aquifoliaceae cuttage seedling method | |
| CN103460854B (en) | A kind of blue or green money willow seed is at southern shandong area germination accelerating method | |
| YODA et al. | Root System Development of Prosopis Deedlings under Different Soil Moisture Conditions | |
| CN104429804A (en) | Big tree transplanting and daily maintenance management method | |
| CN103960030A (en) | Method for cultivating anoectochilus formosanus in wild imitation mode | |
| CN103719176A (en) | Plant antifreezing solution | |
| CN105028156A (en) | Dendrobium officinale tissue culture seedling hardening method | |
| CN103597965B (en) | Method for long-term storage of agate curcuma zedoary seedballs | |
| CN107853293A (en) | A kind of radix ampelopsis seed cryopreservation method | |
| Sahu et al. | Effect of in-situ moisture conservation, forchlorfenuron and boron on growth, fruit cracking and yield of pomegranate cv. Kandhari under rainfed conditions of Himachal Pradesh | |
| CN105475133B (en) | The method that programmed cooling method and Vitrification are combined cryopreservation Eriobotrya germplasm | |
| CN102258010A (en) | Slow refrigerating preservation method for paris polyphylla rootstock | |
| Valkonen et al. | Frost hardiness of Scots pine seedlings during dormancy | |
| KR101412055B1 (en) | Method of germination induction in Pruns yedoensis seeds using liquid nitrogen | |
| CN104255527A (en) | Quick propagation method for regeneration of zanthoxylum molle plants | |
| Afolayan et al. | Germination in Helichrysum aureonitens (Asteraceae): effects of temperature, light, gibberellic acid, scarification and smoke extract | |
| CN107960251A (en) | A kind of Common zenia germ plasm resource strange land live body preserves new method | |
| CN107810949A (en) | A kind of Seed of Platycodon Grandiflorum cryopreservation method | |
| CN105746496A (en) | Method for in-vitro conservation of almond dormant buds by means of vitrification ultralow temperature | |
| CN101530054B (en) | Method for transplanting wile big exbucklandia populnea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180330 |