CN107996562A - A kind of notopterygium root seed cryopreservation method - Google Patents
A kind of notopterygium root seed cryopreservation method Download PDFInfo
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- CN107996562A CN107996562A CN201711312941.9A CN201711312941A CN107996562A CN 107996562 A CN107996562 A CN 107996562A CN 201711312941 A CN201711312941 A CN 201711312941A CN 107996562 A CN107996562 A CN 107996562A
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- notopterygium root
- cooling box
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- 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
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- General Health & Medical Sciences (AREA)
- Agronomy & Crop Science (AREA)
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Abstract
The present invention provides a kind of notopterygium root seed cryopreservation method, including below step：Notopterygium root seed is subjected to 10~30min of seed soaking with 1~3% carboxymethylcellulose sodium solution, is handled with liquid medium, after handling 20~60min, takes out processed notopterygium root seed；Processed notopterygium root seed is put into cryopreservation tube, adds 10~20min of incubation at room temperature of frozen stock solution, then is put it into gradient cooling box；Gradient cooling box with notopterygium root seed is put into low temperature refrigerator, making the temperature of its cooling box, cooling rate is -1 DEG C/min, then takes out cooling box, this cooling box is put into liquid nitrogen at once at -45 DEG C~-60 DEG C, carries out liquid nitrogen cryogenics and saves backup.
Description
Technical field
The present invention relates to a kind of cryopreservation method of notopterygium root seed, belong to the technical field of Germ-plasma resources protection.
Background technology
Notopterygium root alias is Qiang is blue or green, shield Qiang envoy, Hu king envoy, Qiang is sliding, moves back wind envoy, black powder, and Umbelliferae, Notopterygium are for many years
Raw herbaceous plant, high up to 120 centimetres, rhizome is sturdy, and stem is upright, cylinder, band purple.Basal leaf and the lower portion of the stem leaf have handle,
Blade three goes out formula three and returns pinnate compound leaf, and edge incises that shape is shallow to be split to pinniform drastic crack；Leaf sheath film quality, grows and embraces stem.Universal umbel,
Side survivor Chang Buyu；Phyllary is linear, caducous；Involucel piece is linear, spends majority, calyx tooth ovate triangle, petal is white, avette
Avette, the flower pesticide yellow to long round shape, ellipse, style base concora crush slightly swell.The mitogenetic long round shape of fruit, blooms July, 8-9 month results.
It is distributed in Shaanxi, China, Sichuan, Gansu, Qinghai, Tibet.It is grown in border and the shrubbery of 2000-4000 meters of height above sea level.This kind of root
Stem and root are medicinal, warm-natured, dispellieg cold and dampness, for catch cold, have a headache lossless, cold-dampness numbness, upper limb rheumatalgia pain.
The top priority of research of fruit germplasm resource is to collect germplasm, securely held germplasm.Germ-plasma resources protection mode is at present
Room temperature, Cord blood notopterygium root seed two ways.Room temperature preserves Chinese herbal medicine seed, and annual preserve just needs Regeneration again,
Because Chinese herbal medicine seed passes through the preservation of 1 year, vigor reduces quickly；Cord blood Chinese herbal medicine seed, in China, establishes state
Family mid-term storehouse and national genebank, the temperature that national middle term storehouse preserves are 4 DEG C or so, and relative humidity is less than 65%, is typically designed
The service life of stored seeds is 5~10 years, and national genebank preserves storehouse for a long time for germplasm, and the temperature in storehouse is -18 DEG C or so, relatively
Humidity be less than 50%, design shelf-life up to more than 20 years, that is to say, that germplasm preserves in long-term storehouse, vigor likewise as when
Between passage reduce, germplasm generally requires renewal breeding after 20 years, be not permanent preservation.
Therefore, for securely held notopterygium root germ plasm resource, research and probe is a kind of safe and preserves notopterygium root 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
The object of the present invention is to provide a kind of ultra-low temperature preservation method for being suitable for notopterygium root seed, this method can supplement now
The deficiency of notopterygium root Germ-plasma resources protection technology, can be notopterygium root Germ-plasma resources protection with the preservation notopterygium root germ plasm resource of long-term safety
One new effective technological approaches is provided.
The object of the present invention is to provide a kind of notopterygium root seed cryopreservation method, including below step：
(1) notopterygium root seed is subjected to 10~30min of seed soaking with 1~3% carboxymethylcellulose sodium solution at room temperature, used
Liquid medium processing, after handling 20~60min, takes out processed notopterygium root seed；
(2) processed notopterygium root seed is put into cryopreservation tube, adds 10~20min of incubation at room temperature of frozen stock solution, then will
It is put into gradient cooling box；
(3) the gradient cooling box with notopterygium root seed is put into low temperature refrigerator, makes the temperature of its cooling box at -45 DEG C
~-60 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, liquid medium refers to B5 liquid mediums described in step (1), also includes 1.5
~2.5mol/L glycerine and 1~1.5mol/L dimethyl sulfoxide (DMSO)s.
The A reagents preferably 110~121 DEG C of more than high pressure steam sterilization 15min, are cooled to room temperature placement.
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, frozen stock solution refers to B5 liquid mediums described in step (2), also include 2.8~
3.8mol/L glycerine, 1.6~2mol/L dimethyl sulfoxide (DMSO)s and 0.45~0.6mol/L sucrose.
The B reagents preferably 110~121 DEG C of more than high pressure steam sterilization 15min, are cooled to room temperature placement.
In a concrete scheme, the temperature requirement of low temperature refrigerator is generally below -70 DEG C described in step (3).
When above-mentioned preserved notopterygium root 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, notopterygium root seed in cryopreservation tube is taken out, then with tap water or distills water washing 3~5 times, notopterygium root after cleaning
Seed is directly used in culture.
Technique effect
1st, the present invention carries out notopterygium root cryopreservation, and technique is simple, and stability is reliable, the germination of the notopterygium root seed after preservation
The germination percentage of rate and fresh kind there was no significant difference notopterygium root.
2nd, the present invention has long-term, effective, securely held notopterygium root seed, and it is most effective to be that notopterygium root germ plasm resource preserves for a long time
Method.
Embodiment
In the following, 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 notopterygium root that Certain District, Hunan harvests then is taken, is dried naturally, by the notopterygium root seed of water content 9.2% or so with 2% carboxylic
Methylcellulose sodium solution carries out 10~30min of seed soaking, with containing 2.2mol/L glycerine and 1.3mol/L dimethyl sulfoxide (DMSO)s
B5 liquid mediums processing, handle 20min after, take out processed notopterygium root seed, place into cryopreservation tube, add B5 liquid
Nutrient solution, wherein also contain 3.2mol/L glycerine, 1.7mol/L dimethyl sulfoxide (DMSO)s and 0.6mol/L sucrose, incubation at room temperature 15min,
Put it into again in gradient cooling box；Notopterygium root puts gradient cooling box into -70 DEG C of low temperature refrigerators and carries out gradient cooling, makes its cooling
For the temperature of box at -50 DEG C, cooling rate is -1 DEG C/min, then takes out cooling box, this cooling box is put into liquid nitrogen at once, into
After row liquid nitrogen cryogenics preserve 8 years, cryopreservation tube is taken out from liquid nitrogen, places room temperature at 23 DEG C or so, it is directly natural using room temperature
Thaw, after having thawed, take out notopterygium root seed in cryopreservation tube, then with tap water or distill water washing 4 times, notopterygium root seed after cleaning
Seed germination is directly used in, germination percentage reaches 96.61% after tested.
Comparative examples 1
The notopterygium root that Certain District, Hunan harvests then is taken, is dried naturally, by the notopterygium root seed of water content 11% or so with 2% carboxylic first
Base sodium cellulosate solution carries out 10~30min of seed soaking, is handled with the B5 liquid mediums containing 2.2mol/L glycerine, processing
After 20min, processed notopterygium root seed is taken out, is placed into cryopreservation tube, B5 liquid mediums are added, wherein also containing
3.2mol/L glycerine, 1.7mol/L dimethyl sulfoxide (DMSO)s and 0.6mol/L sucrose, then put it at once in 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, cooling rate for -1 DEG C/
Min, then takes out cooling box, this cooling box is put into liquid nitrogen at once, carry out liquid nitrogen cryogenics preserve 8 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 notopterygium root in cryopreservation tube
Seed, then with tap water or distill water washing 4 times, notopterygium root seed is directly used in Seed germination after cleaning, after tested germination percentage
Reach 76.52%.
Comparative examples 2
The notopterygium root that Certain District, Hunan harvests then is taken, is dried naturally, by the notopterygium root seed of water content 11% or so with 2% carboxylic first
Base sodium cellulosate solution carries out 10~30min of seed soaking, with containing 2.2mol/L glycerine and 1.3mol/L dimethyl sulfoxide (DMSO)s
The processing of B5 liquid mediums, after handling 20min, takes out processed notopterygium root seed, places into cryopreservation tube, adds the training of B5 liquid
Nutrient solution, wherein also contain 3.2mol/L glycerine, 1.7mol/L dimethyl sulfoxide (DMSO)s and 0.6mol/L sucrose, incubation at room temperature 15min, then
Put it into after -5 DEG C or so low temperature refrigerators preserve 8 years, cryopreservation tube taken out from liquid nitrogen, place room temperature at 23 DEG C or so,
Room temperature naturally to thaw is directly utilized, after having thawed, takes out notopterygium root seed in cryopreservation tube, then with tap water or distill water washing 4
Secondary, notopterygium root seed is directly used in Seed germination after cleaning, and germination percentage reaches 41.25% after tested.And take fresh 1~5 DEG C of notopterygium root
Lower Cord blood is after 8 years, and for Seed germination, notopterygium root fails to germinate after tested.
Embodiment 2
The notopterygium root that Certain District, Hunan harvests then is taken, is dried naturally, by the notopterygium root seed of water content 6% or so with 3% carboxylic first
Base sodium cellulosate solution carries out 10~30min of seed soaking, with the B5 containing 2mol/L glycerine and 1.5mol/L dimethyl sulfoxide (DMSO)s
Liquid medium processing, after handling 30min, takes out processed notopterygium root seed, places into cryopreservation tube, adds B5 Liquid Cultures
Liquid, wherein also contain 3mol/L glycerine, 1.8mol/L dimethyl sulfoxide (DMSO)s and 0.5mol/L sucrose, incubation at room temperature 20min, then by its
It is put into gradient cooling box；Put gradient cooling box into -80 DEG C of low temperature refrigerators and carry out gradient cooling, make the temperature of its cooling box
At -60 DEG C or so, 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
After nitrogen Cord blood 10 years, cryopreservation tube is taken out from liquid nitrogen, room temperature is placed at 23 DEG C or so, is directly solved naturally using room temperature
Freeze, after having thawed, take out notopterygium root seed in cryopreservation tube, then with tap water or distill water washing 4 times, notopterygium root seed is straight after cleaning
Connect and be used for Seed germination, germination percentage reaches 97.17% after tested.
Claims (6)
1. a kind of notopterygium root seed cryopreservation method, including below step：
(1) notopterygium root seed is subjected to 10~30min of seed soaking with 1~3% carboxymethylcellulose sodium solution at room temperature, uses liquid
Nutrient solution processing, after handling 20~60min, takes out processed notopterygium root seed；
(2) processed notopterygium root seed is put into cryopreservation tube, adds 10~20min of incubation at room temperature of frozen stock solution, then is put
Enter in gradient cooling box；
(3) the gradient cooling box with notopterygium root seed is put into low temperature refrigerator, makes the temperature of its cooling box -45 DEG C~-60
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 spare.
2. the method according to claim 1, wherein step (1) the A reagents refer to B5 liquid mediums, also include 1.5~
2.5mol/L glycerine and 1~1.5mol/L dimethyl sulfoxide (DMSO)s.
3. the method according to claim 1, wherein the A reagents preferably 110~121 DEG C of more than high pressure steam sterilization 15min, cold
But placed to room temperature.
4. contain 100% isopropanol or glycerine in the method according to claim 1, wherein step (2) the gradient cooling box, can
To reuse.
5. the method according to claim 1, wherein step (2) the B reagents refer to B5 liquid mediums, also include 2.8~
3.8mol/L glycerine, 1.6~2mol/L dimethyl sulfoxide (DMSO)s and 0.45~0.6mol/L sucrose.
6. the method according to claim 1, wherein the B reagents preferably 110~121 DEG C of more than high pressure steam sterilization 15min, cold
But placed to room temperature.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711312941.9A CN107996562A (en) | 2017-12-11 | 2017-12-11 | A kind of notopterygium root seed cryopreservation method |
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| CN201711312941.9A CN107996562A (en) | 2017-12-11 | 2017-12-11 | A kind of notopterygium root seed cryopreservation method |
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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
- 2017-12-11 CN CN201711312941.9A patent/CN107996562A/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 (5)
| 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 * |
| 王蒂 等: "《细胞工程学》", 31 July 2003, 中国农业出版社 * |
| 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 * |
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Application publication date: 20180508 |