CN106105586A - A kind of method of Cryopreservation Radix Angelicae Dahuricae seed - Google Patents
A kind of method of Cryopreservation Radix Angelicae Dahuricae seed Download PDFInfo
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- CN106105586A CN106105586A CN201610475730.6A CN201610475730A CN106105586A CN 106105586 A CN106105586 A CN 106105586A CN 201610475730 A CN201610475730 A CN 201610475730A CN 106105586 A CN106105586 A CN 106105586A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01F—PROCESSING OF HARVESTED PRODUCE; HAY OR STRAW PRESSES; DEVICES FOR STORING AGRICULTURAL OR HORTICULTURAL PRODUCE
- A01F25/00—Storing agricultural or horticultural produce; Hanging-up harvested fruit
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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Abstract
A kind of method that the invention discloses Cryopreservation Radix Angelicae Dahuricae seed.It comprises the steps: 1) by Radix Angelicae Dahuricae seed drying, obtain the seed being dried；2) described dry seed is packed, keep in cold storage, obtain the seed of freezing；3) seed to described freezing thaws and stores.The present invention is simple to the storage method of Radix Angelicae Dahuricae seed；The time of storage is long；Little on Radix Angelicae Dahuricae percentage of seedgermination, electrical conductivity, peroxidase (POD) activity and content of soluble protein impact.
Description
Technical field
A kind of method that the present invention relates to Cryopreservation Radix Angelicae Dahuricae seed, belongs to Chinese crude drug the storage of seeds field.
Background technology
The Radix Angelicae Dahuricae is the samphire Radix Angelicae Dahuricae (Angelica dahurica (Fisch.ex Hoffm.) Benth.et
Or Radix angelicae dahuricae (Angelica dahurica (Fisch.ex Hoffm.) Benth.et Hook.f)
Hook.f.var.formosana (Boiss.) Shan et Yuan) dry root, because primary root bar is the root of Dahurian angelica, color is white, therefore named in vain
The root of Dahurian angelica, is used as medicine with root, and it is warm in nature, gas fragrance, and acrid in the mouth, micro-hardship are returned stomach, colon, lung meridian, had the dehumidifying of scattered wind, sensible analgesia, detumescence
The effects such as evacuation of pus, can medicine-food two-purpose.According to statistics, the prescribed preparation of Radix Angelicae Dahuricae composition has more than 600 to plant, and occupies importantly in Chinese crude drug
Position.Successive dynasties recipe such as Notoptreygium Decoction of Nine Ingredients, huoxiang zhengqi powder etc. all have it to be used as medicine.The Radix Angelicae Dahuricae is Genuine crude drugs in Sichuan, and main product is in Sichuan
Suining, its yield is high, accounts for whole nation Radix Angelicae Dahuricae total output more than 70%, in addition to supply domestic market, also finds a good sale in Hong Kong, Southeast Asia, day
The ground such as basis and America and Europe.
The Radix Angelicae Dahuricae, based on seminal propagation, uses culturing and transplanting seedlings more, and under routine preservation means, next year aged seed germination percentage is not
Height, and the reason restricting percentage of seedgermination is unclear.Generally, Radix Angelicae Dahuricae seed longeivity about 1 year, seed is good and bad to field
Emergence rate, yield, quality have significant impact, thus affect the income at medical material plantation family.Therefore, the cultivation seed of angelica root is
Crucial.
Summary of the invention
A kind of method that it is an object of the invention to provide Cryopreservation Radix Angelicae Dahuricae seed, the present invention storage to Radix Angelicae Dahuricae seed
Time is long.
The method of the Cryopreservation Radix Angelicae Dahuricae seed that the present invention provides, comprises the steps: 1) by Radix Angelicae Dahuricae seed drying,
To the seed being dried；
2) described dry seed is packed, keep in cold storage, obtain the seed of freezing；
3) seed to described freezing thaws and stores.
In above-mentioned method, step 2) in also include the step that mixed by described dry seed with anti-frost protection agent.
In above-mentioned method, described dry seed is 100:5～10 with the mass ratio of described anti-frost protection agent, specifically may be used
For 100:5 or 100:10；
Described anti-frost protection agent includes at least one in dimethyl sulfoxide, glycerol and Polyethylene Glycol (being called for short PEG)；Described
The molecular weight of Polyethylene Glycol concretely 400.
In above-mentioned method, the water content of described dry seed can be 5～10%, concretely 5.34%, 7.70%,
9.43% or 5.34～9.43%, most preferably 9.43%；Concrete dry run, for using 105 DEG C of low constant temperature, dries 4h, it is thus achieved that
Radix Angelicae Dahuricae seed moisture content, seed is put in the exsiccator with silica gel as desiccant, is dried through different time, and seed moisture content regulates extremely
Required water content.
In the present invention, the water content of described dry seed is according to mass water content meter.
In above-mentioned method, the material packing described dry seed includes plastic centrifuge tube, plastics valve bag and Corii Bovis seu Bubali
At least one in paper；
The thickness of the material packing described dry seed can be 0.1～1mm.
In the present invention, described dry seed airtight package, the described plastic centrifuge tube of employing is centrifuge tube with cover；Mould
The thickness concretely 0.1mm of material valve bag.
In above-mentioned method, described in keep in cold storage and include quick freezing mode, slow freezing mode gentle speed freezing mode
In at least one；
Described quick freezing mode is freezing 0～60d in liquid nitrogen；
Described slow freezing mode is in 1～10 DEG C of freezing 0.2～1h, then-25～-15 DEG C of freezings 4～5h, finally
It is positioned in described liquid nitrogen freezing 0～60d；Concretely at 5 DEG C of freezing 0.5h, then at-20 DEG C of freezing 4h, finally it is positioned over
Freezing 0～60d in described liquid nitrogen；
Described slow freezing mode is in 1～10 DEG C of freezing 0.2～1h, then-25～-15 DEG C of freezings 0.5～1h,
After be positioned in described liquid nitrogen freezing 0～60d；Concretely at 5 DEG C of freezing 0.5h, then at-20 DEG C of freezing 1h, finally place
Freezing 0～60d in described liquid nitrogen.
In the present invention, the temperature in described liquid nitrogen environment refers to-196 DEG C.
In above-mentioned method, described defrosting storage includes quick-thawing mode, at a slow speed thawing mode and slow thawing mode
In at least one；
Described quick-thawing mode is for moving to thaw in 30～50 DEG C from described liquid nitrogen；Concretely from described liquid nitrogen
In move to 40 DEG C of warm water and thaw；
Described thawing mode at a slow speed for move to successively from described liquid nitrogen-25～-15 DEG C thaw 0.5～2h, 3～8 DEG C of solutions
Freeze；From described liquid nitrogen, concretely move to-20 DEG C of defrosting 1h, 4 DEG C of defrostings successively；
Described slow thawing mode is for move to 15～25 DEG C of defrostings from described liquid nitrogen.
The invention have the advantages that
The present invention is simple to the storage method of Radix Angelicae Dahuricae seed；The time of storage is long；To Radix Angelicae Dahuricae percentage of seedgermination, conductance
Rate, peroxidase (POD) activity and content of soluble protein impact are little.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
In following embodiment, for the examination Radix Angelicae Dahuricae (Angelica dahurica) seed source in Yongxing town, Sichuan Suining City
Ridge village, gathers for 2014；
The mensuration of water content, and reference article (Yang Zhizhong, Jiang Yunbin, Mu Xiangrong etc. angelica anomala seed testing method is ground
Study carefully [J]. seed, 2013,3 (12): 119～122.) in method；Detailed process, for using 105 DEG C of low constant temperature, is dried 4h, is obtained
Obtain Radix Angelicae Dahuricae seed moisture content；Seed is put in the exsiccator with silica gel as desiccant, is dried through different time, and seed moisture content regulates
To required water content.
Embodiment 1, the storage of Radix Angelicae Dahuricae seed
1) Radix Angelicae Dahuricae seed drying, moisture regulation to 5.34%, 7.70% and 9.43% 3 kind of different moisture content, then will not
Put in sealed aluminum box standby with the seed of water content.
2) by above-mentioned 1) in the Radix Angelicae Dahuricae seed of different moisture content, respectively with dimethyl sulfoxide, glycerol, PEG400
(PEG400) as anti-frost protection agent mixing (Radix Angelicae Dahuricae seed is as shown in table 1 with the mass ratio of anti-frost protection agent), the most respectively
It is contained in plastic centrifuge tube (with cover), plastics valve bag (0.1mm is thick), is respectively adopted following three kinds of modes and keeps in cold storage:
Quick freezing: liquid nitrogen；
Slow freezing: refrigerator (5 DEG C, 0.5h)-refrigerator (-20 DEG C, 4h)-liquid nitrogen；
Slow freezing: refrigerator (5 DEG C, 0.5h)-refrigerator (-20 DEG C, 1h)-liquid nitrogen.
3) by above-mentioned 2) in freezing seed, be respectively adopted following three kinds of modes and thaw storage:
Quick-thawing: liquid nitrogen moves to 40 DEG C of warm water and thaws；
Thaw at a slow speed: liquid nitrogen-refrigerator (-20 DEG C, 1h)-refrigerator (4 DEG C) thaws；
Slow defrosting: liquid nitrogen moves to 20 DEG C of environment and thaws.
Above-mentioned 2) time preserved in liquid nitrogen in-3) is 7d, 30d or 60d, the most as shown in table 1.
Above-mentioned condition uses Orthogonal Experiment and Design to carry out the optimization of preferred plan, obtains optimal seed ultra-low temperature storage
Combination；Select L18 (21+37) orthogonal table.Each factor of orthogonal test and level thereof are shown in Table 1.According to Orthogonal Experiment and Design, enter altogether
18 tests of row, test the seed not carrying out Cryopreservation under 4 DEG C of environment for comparison.
Table 1 orthogonal test column table
From in table 1, the packaging material that the present invention selects are plastic centrifuge tube and 0.1mm plastics valve bag, and seed contains
It is 5.34%, 7.70% and 9.43% respectively that the water yield arranges 3 levels, and protective agent dimethyl sulfoxide, glycerol, PEG set respectively
Setting to 0 %, 5% and 10%3 levels, freezing mode, thawing mode are divided into 3 class fast, slow, slow, and cold preservation time arranges 7
My god, 30 days, 60 days 3 levels.
After above-mentioned storage method, Radix Angelicae Dahuricae seed activity determination experiment is measured according to the method for following 1-5:
1, seed germination experiment
Incubator arranges 25 DEG C/18 DEG C alternating temperatures round the clock, illumination 12h, and every kind of process random number of every part of material takes 100 kinds
Son, repeats for 3 times.
(n is final germinative number to germination percentage (GR) (%)=n/N × 100%；N is for for planting experimentally subnumber).
It is calculated as germinateing when the plumular axis length breaking through seed coat reaches pure dan its length, and as first gate time.
Seed germination reaches the highest, and the natural law without new germination seed is last gate time the most again.
2, conductance measurement
Random number takes 50 clean seeds of Radix Angelicae Dahuricae seed, repeats for 3 times.Rinse 3 times with ionized water, blot surface water with filter paper
Point, seed is put in 250mL beaker, add 100mL deionized water, measure initial conductivity；With preservative film, beaker is sealed,
Seed leachate electrical conductivity is measured after standing 24h under room temperature condition.
Seed leachate electrical conductivity [μ s/ (cm*g)]=(seed leachate electrical conductivity-initial conductivity)/seed sample matter
Amount.
3, peroxidase (POD) activity
Take Radix Angelicae Dahuricae seed 0.5g, be placed in mortar, add 5ml 0.02mol/L KH2PO4Grind to form homogenate, 4000r/
Min, centrifugal 15min, collect supernatant and be saved on ice.Residue obtained again with 5ml 0.02mol/L KH2PO4Extract once, close
And twice supernatant, zyme extract 10ml ice bath grinds, is centrifuged 15min at 4 DEG C of 1200r/min, and supernatant is enzyme extract,
Preserve at 0～4 DEG C.
Take zyme extract 0.5ml (diluting stock solution depending on enzymatic activity size), add POD reactant liquor 3ml, include 0.08%H2O2,
The PBS (pH 7.0) of 0.1% guaiacol, colorimetric at 470nm immediately, every 30s records single reading, records reading 3min,
Taking and calculate the change of OD value in line part, unit of enzyme activity represents with △ OD 470/min g.
Reaction mixture configuration 100mmol/L pH 6.0 phosphate buffer 50ml, adds guaiacol 28ul, in magnetic force
Heated and stirred on agitator, until guaiacol dissolves, after solution cools down, adds 30% hydrogen peroxide 19ul, mix homogeneously,
It is stored in refrigerator, standby.
4, content of soluble protein measures
Method with reference to Gao Junfeng[8]Measure content of soluble protein in Radix Angelicae Dahuricae seed, slightly change.
Take Radix Angelicae Dahuricae seed 0.5g, be placed in mortar after grinding to form homogenate with pH 7.0 phosphate buffer, constant volume 25ml,
10000r/min is centrifuged 10min, takes supernatant 1ml (opsin's matter content suitably dilutes) in test tube, standby.
Pipette samples extracting solution 0.1ml, puts in tool plug scale test tube and (sets two and repeat pipe), add 5ml coomassie bright
Blue G-250 reagent, is sufficiently mixed, and places after 2min colorimetric under 595nm, records absorbance, by standard curve recurrence side
Journey Y=0.0608x+0.2218 (R2=0.9073) protein content is checked in.
Protein content (%)=C × V2 × V0×100/(m×V1)
In formula: the protein concentration that C checks in from standard curve, g/mL
V0The cumulative volume of sample solution, mL
Sample-adding amount when V1 measures, mL
Constant volume when V2 measures, mL
The quality of m sample, g
5, Data Analysis Services
Use DPS statistical analysis software that data carry out variance analysis, use duncan's new multiple range method to carry out significance analysis.Real
Test result:
Shown in following (1)-(4) of the experimental result of above-mentioned 1-5.
(1) Excised Embryos impact on Radix Angelicae Dahuricae germination
Table 2 Orthogonal Experiment and Design L18 (21+37) and percentage of seedgermination
In table 2, A: packaging material；B: water content；C: dimethyl sulfoxide；D: glycerol；E:PEG；F: freezing mode；G: thaw
Mode；H: storage time；M: average；R: extreme difference.
Table 3 germination test analysis of variance table
In table 3, MS: sum of square of deviations；Df: degree of freedom.
Table 4 Radix Angelicae Dahuricae seed Cryopreservation important factor in order synopsis
Table 2 is applied orthogonal design L18 (21+37), factor A, B, C, D, E, F, G and H be respectively packaging material, water content,
Dimethyl sulfoxide, glycerol, PEG400, freezing mode, thawing mode and storage time, all according to test bar in table 1 in embodiment 1
Part is carried out.A factor is 2 levels, 3 levels of remaining selecting factors, and each level is made 3 times and repeated, and result is as shown in table 2.
From Table 2, it can be seen that in each factor, Radix Angelicae Dahuricae germination is affected maximum is freezing mode, next to that solve
Freeze mode.The order that 8 factors affect Radix Angelicae Dahuricae percentage of seedgermination is: F > G > A > D > C > H > B > E.Variance analysis shows, freezing side
Formula, thawing mode energy pole appreciable impact Radix Angelicae Dahuricae germination, packaging material energy appreciable impact germination, remaining 5 factor pair
The impact of germination is not notable (as shown in table 3), but it is discrepant for having between the varying level of same factor.It addition, bag
When package material uses plastic centrifuge tube, the germination percentage of Radix Angelicae Dahuricae seed is higher, is 43.19%.Quick freezing and slow freezing are respectively
Percentage of seedgermination the highest (52.45%) and two kinds of freezing modes of minimum (32.95%), extreme difference is 19.50%.At thawing mode
In when thawing at a slow speed percentage of seedgermination the highest, be 45.5%, during quick-thawing, germination percentage is minimum, is 39.28% (such as table 4 institute
Show).
(2) Excised Embryos impact on Radix Angelicae Dahuricae Conductivity
The Radix Angelicae Dahuricae seed processing application orthogonal design carries out conductance measurement, and generally, seed vitality is the lowest, electricity
Conductance is the highest.From table 2 it can be seen that Radix Angelicae Dahuricae seed is in Cryopreservation, leachate electrical conductivity is affected by each factor
Maximum is freezing mode, next to that thawing mode.8 factors affect the order of Radix Angelicae Dahuricae Conductivity successively: F > G > E > D
>C>A>H>B.Variance analysis shows, freezing mode energy pole appreciable impact Radix Angelicae Dahuricae seed leachate electrical conductivity, PEG, thawing mode pair
The impact of Radix Angelicae Dahuricae seed leachate electrical conductivity is significant difference, the impact of other 7 factor pair Radix Angelicae Dahuricae seed leachate electrical conductivity
Not notable (being shown in Table 3).It addition, leachate electrical conductivity is minimum in 3 process levels when the volume fraction of PEG is 10%, for
17.79μs/cm*g.In 3 kinds of freezing modes, the effect of quick freezing is best, and leachate electrical conductivity is minimum, is 15.73 μ s/
Cm*g, the effect of slow freezing is worst, and leachate electrical conductivity is the highest, is 20.52 μ s/cm*g, and the two extreme difference is 4.79 μ s/cm*
g.Effect in 3 kinds of thawing modes of thawing at a slow speed is best, and leachate electrical conductivity is less than comparison (being shown in Table 4).
(3) Excised Embryos impact on peroxidase (POD) activity
Peroxidase is one of intracellular critically important degeneration-resistant enzyme, weighs seed vitality, high vigor kind by POD activity
Sub-resistance enzyme activity is strong.As shown in Table 2, each factor is freezing mode to Radix Angelicae Dahuricae seed POD activity influence maximum, next to that
Thawing mode.8 factors affect the order of Radix Angelicae Dahuricae seed POD activity successively: F > G > C > A > D > E > H > B.Variance analysis shows,
Freezing mode energy pole appreciable impact POD activity, the impact of other 7 factor pair POD activity is the most notable.It addition, at 3 freezing modes
In, the effect of quick freezing is best, POD activity the highest 16.59U g-1min-1, the effect of slow freezing is worst, and POD activity is
Low 13.44U g-1min-1, less than comparison 14.33U g-1min-1。
(4) Excised Embryos impact on soluble protein
In seed, soluble protein is the enzyme participating in various metabolism mostly, and its content height is all kinds of in reflecting plant
The activity level of enzyme, has certain dependency with seed vitality, and seed vitality reduces its soluble protein content and declines.By table 2
Understanding, each factor is freezing mode on Radix Angelicae Dahuricae Seed Soluble Protein content impact maximum, next to that PEG.8 factor impacts
The order of Radix Angelicae Dahuricae Seed Soluble Protein content is successively: F > E > G > A > C > B > D=H.Variance analysis shows, freezing mode is conciliate
Freezing mode energy pole appreciable impact soluble protein content, PEG is significant difference to soluble protein content, other 5 factor pairs
The impact of soluble protein content is the most notable.It addition, along with PEG volume fraction increase soluble protein content constantly on
Rise, but be below comparison.In 3 kinds of freezing modes, the effect of quick freezing is best, and soluble protein content is the highest, for
6.92mg/g, higher than the 5.62mg/g of comparison, during slow freezing mode, soluble protein is minimum, for 4.09mg/g.Thaw at a slow speed
In 3 kinds of thawing modes, the best soluble protein content of effect is the highest, and for 5.78mg/g, during quick-thawing, soluble protein is
Low, for 5.11mg/g.
Above-mentioned result of the test shows, Radix Angelicae Dahuricae seed of the present invention can be preserved in ultralow temperature mode, in 9.43% water content
Radix Angelicae Dahuricae seed plastic centrifuge tube as packaging material, under the protection of 10%PEG anti-frost protection agent, use immediate mode cold
Freeze, after Liquid nitrogen, thaw with slow mode, the electrical conductivity of seed can be effectively reduced, improve the germination of seed
Rate, POD activity and soluble protein content, delay Seed senescence.The optimization process of the present invention is combined as: plastic centrifuge tube+
9.43% water content+10%PEG+ quick freezing+thaw at a slow speed.
Claims (7)
1. a method for Cryopreservation Radix Angelicae Dahuricae seed, comprises the steps: 1) by Radix Angelicae Dahuricae seed drying, obtain the kind being dried
Son；
2) described dry seed is packed, keep in cold storage, obtain the seed of freezing；
3) seed to described freezing thaws and stores.
Method the most according to claim 1, it is characterised in that: in described method, step 2) in also include being dried described
The step that mixes with anti-frost protection agent of seed.
Method the most according to claim 1 and 2, it is characterised in that: described dry seed and described anti-frost protection agent
Mass ratio is 100:5～10；
Described anti-frost protection agent includes at least one in dimethyl sulfoxide, glycerol and Polyethylene Glycol.
4. according to the method according to any one of claim 1-3, it is characterised in that: the water content of described dry seed is 5
～10%.
5. according to the method according to any one of claim 1-4, it is characterised in that: pack the material bag of described dry seed
Include at least one in plastic centrifuge tube, plastics valve bag and kraft paper；
The thickness of the material packing described dry seed is 0.1～1mm.
6. according to the method according to any one of claim 1-5, it is characterised in that keep in cold storage described in: and include quick freezing side
At least one in formula, slow freezing mode gentle speed freezing mode；
Described quick freezing mode is freezing 0～60 day in liquid nitrogen；
Described slow freezing mode is in 1～10 DEG C of freezing 0.2～1h, then-25～-15 DEG C of freezings 4～5h, finally places
Freezing 0～60d in described liquid nitrogen；
Described slow freezing mode is in 1～10 DEG C of freezing 0.2～1h, then-25～-15 DEG C of freezings 0.5～1h, finally
It is positioned in described liquid nitrogen freezing 0～60d.
7. according to the method according to any one of claim 1-6, it is characterised in that: described defrosting storage includes quick-thawing side
At least one in formula, at a slow speed thawing mode and slowly thawing mode；
Described quick-thawing mode is for moving to thaw in 30～50 DEG C from described liquid nitrogen；
Described thawing mode at a slow speed for move to successively from described liquid nitrogen-25～-15 DEG C thaw 0.5～2h, 3～8 DEG C of defrostings；
Described slow thawing mode is for move to 15～25 DEG C of defrostings from described liquid nitrogen.
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| CN110278735A (en) * | 2019-04-25 | 2019-09-27 | 成都中医药大学 | A kind of Semen Carthami of cryopreservation goes out storage method |
| CN110278735B (en) * | 2019-04-25 | 2022-01-04 | 成都中医药大学 | Method for warehousing and warehousing safflower seeds stored at low temperature |
| CN113466470A (en) * | 2021-07-26 | 2021-10-01 | 四川农业大学 | Response surface method optimized optimal buffer solution extraction test method for germinating seed protein |
| CN113466470B (en) * | 2021-07-26 | 2023-07-25 | 四川农业大学 | Optimal buffer solution extraction test method for optimizing sprouted seed protein by response surface method |
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