CN110278735A - A kind of Semen Carthami of cryopreservation goes out storage method - Google Patents
A kind of Semen Carthami of cryopreservation goes out storage method Download PDFInfo
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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
The present invention provides a kind of Semen Carthami cryopreservations to go out to be put in storage freeze-thaw method, it is after freezing Semen Carthami, it is stored in the seed bank of different temperatures respectively, it freezes, it thaws when outbound, concrete operations are as follows: 1) mid-term library: Semen Carthami is placed in -4 ± 2 DEG C of seed banks, when outbound, and gradient increased temperature to room temperature；2) long-term library: carrying out gradient cooling for Semen Carthami, then is placed in -18 ± 2 DEG C of seed banks, when outbound, gradient increased temperature to room temperature；3) ultralow cold storage: Semen Carthami being placed in liquid nitrogen container cooling, then is placed in -75 ± 5 DEG C of seed banks, when outbound, is placed in tepidarium, is thawed.Cryopreservation through the invention goes out to be put in storage freeze-thaw method, and obtained Semen Carthami germination percentage is high, and SOD activity and relative conductivity are lower, POD and CAT activity is then higher, and the content of reserve substance is also high in seed, has application value.
Description
Technical field
Present invention relates particularly to a kind of Semen Carthamis of cryopreservation to go out storage method.
Background technique
Semen Carthami originates from Mediterranean east bank from herbaceous plant safflower (Carthamus tinctorius L.)
Crescent.Semen Carthami is achene, and ellipse or obovate are about 5 millimeters, and base portion is slightly crooked, white, no pappus, tool four
Rib.Chinese medicine is entitled " SEMEN CARTHAMI ".33.6-49.8 grams of mass of 1000 kernel.Outer layer is the thick white protective shell of comparison, the light that this shell has
Sliding, some is in carinate.Adhere to one layer thin of kind skin in shell, embryo is enclosed in kind of intradermal and protein contained in embryo.The flower of safflower
Labeled with white, yellow, orange, pink, scarlet and peony.Safflower is other than its seed is used as oil expression, flower, pollen etc.
Also tool has been widely used, and has blood circulation, removing blood stasis and acesodyne, anticancer, extracts pigment, beautifying and antisenility and wait for a long time multiple functions.It is red
Inherently a kind of parts of generic medicinal plants of flower, Compendium of Material Medica claim it " can invigorate blood circulation, moisturize, relieve pain, dissipate swollen, promoting menstruation ".
Medicinal Plant Germplasm Resources are the bases of traditional Chinese medicine development, are new Chinese medicine, botanical medicine exploitation, excellent product
The gene source of kind breeding.Plant germplasm resource library refers to the preservation facility using plant germplasm resource as protected object, and low temperature
Germplasm resource bank is the optimal path for saving plant germplasm resource, but China professional Chinese medicine (medicinal plant) germ plasm resource is low
Lag is compared in warm library building-up work, is the preservation system for using for reference crop genetic resource library mostly, so for the low temperature of medicinal plant
Preservation system is simultaneously not perfect, this just carrys out certain difficulty in the permanently effective work belt that saves of Freezing room to germplasm.
The Germplasm Resources of Farm Crop material point two-stage of China saves.The store materials of long-term preservation are protected in long-term library, mid-term
The material preservation deposited is in mid-term library, and the agricultural sciences worker of some countries is committed to storing using ultralow cold storage, to reach
To the purpose of long-term storage germplasm.And there is not the research report for the cryo-conservation system of Semen Carthami also at present.
Summary of the invention
To solve the above problems, it is an object of that present invention to provide a kind of mid-term library, (- 4 ± 2 DEG C of seed banks, storage period are reachable
15 years), long-term library (- 18 ± 2 DEG C of seed banks, storage period was up to 50 years), ultralow cold storage (- 75 ± 5 DEG C of seed banks, it is long-term to store
Deposit) the lower Semen Carthami of storage is put in storage and method of disposal when outbound, and protect the germination percentage of Semen Carthami and vigor.
The present invention provides a kind of Semen Carthamis of cryopreservation to go out storage method, the method is as follows:
1) when being stored in mid-term library: Semen Carthami is placed directly within the storage of -4 ± 2 DEG C of seed banks, when outbound, gradient increased temperature
To room temperature；
2) when being stored in long-term library: Semen Carthami is first stood at 2~8 DEG C, then is stored in -18 ± 2 DEG C of seed banks, out
When library, gradient increased temperature to room temperature；
3) when being stored in ultralow cold storage: Semen Carthami is first placed in liquid nitrogen and freezes, then is stored in -75 ± 5 DEG C of seeds
Library when outbound, is first placed in tepidarium and thaws, then be placed at room temperature for；
The gradient increased temperature to room temperature is first 2~8 DEG C of standings, then tepidarium, is finally placed at room temperature for.
Further, step 1) and the step 2) Semen Carthami are to heat sealed package with two layers of aluminum foil compound bag.
Further, 4 DEG C, 24~30h of time of the step 2) dwell temperature, preferably for 24 hours.
Further, the step 3) Semen Carthami is loaded in cryopreservation tube.
Further, step 3) is described is placed in 5~10min of liquid nitrogen time, preferably 5min.
Further, the temperature of the step 3) tepidarium is 40 ± 2 DEG C, 5~10min of time, preferably 5min.
Further, the gradient increased temperature to room temperature is first 2~8 DEG C of 5~10min of standing, then 40 ± 2 DEG C of water-baths 5~
10min is finally placed at room temperature for.
Further, the gradient increased temperature to room temperature is first 4 DEG C of standing 5min, then 40 ± 2 DEG C of water-bath 5min, finally
It is placed at room temperature for.
Semen Carthami cryopreservation of the invention goes out to be put in storage freeze-thaw method, to the freezing side before Semen Carthami storage
Thawing mode when formula and outbound has done detailed elaboration.Cryopreservation through the invention goes out to be put in storage freeze-thaw method,
Obtained Semen Carthami germination percentage is high, and SOD activity and relative conductivity are lower, POD and CAT activity is then higher, storage in seed
The content of substance is high, has application value.
Obviously, above content according to the present invention is not taking off according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes above content of the invention further detailed
Explanation.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all above-mentioned interior based on the present invention
Hold realized technology to all belong to the scope of the present invention.
Detailed description of the invention
The germination percentage variation tendency of Semen Carthami under Fig. 1 difference preservation condition
The relative conductivity variation tendency of Fig. 2 difference freezing and each temperature Semen Carthami that thaws
The soluble sugar (A) and soluble protein (B) changes of contents of Fig. 3 difference freezing and each temperature Semen Carthami that thaws
Trend
Semen Carthami activities of antioxidant enzymes variation tendency under Fig. 4 difference preservation condition
Specific embodiment
The Semen Carthami cryopreservation of the present invention of embodiment 1 goes out to be put in storage freeze-thaw method
1) mid-term library: two layers of aluminum foil compound bag of Semen Carthami is heated into sealed package, 4 ± 2 DEG C of seed banks is placed in and protects
It deposits, when outbound, first 4 DEG C of standing 5min, then 40 ± 2 DEG C of water-bath 5min are finally placed at room temperature for；
2) it long-term library: after two layers of aluminum foil compound bag heating sealing of Semen Carthami, is placed for 24 hours in 4 DEG C of refrigerators, then set
When -18 ± 2 DEG C of seed banks preservations, outbound, first 4 DEG C of standing 5min, then 40 ± 2 DEG C of water-bath 5min are finally placed at room temperature for；
3) ultralow cold storage: Semen Carthami is loaded in cryopreservation tube, is placed in the cooling 5min of liquid nitrogen container, then be placed in -75 ± 5 DEG C
Seed bank saves, and when outbound, is placed in 40 ± 2 DEG C of tepidariums the 5min that thaws, is finally placed at room temperature for.
Beneficial effects of the present invention are illustrated below by way of test example:
Test example 1
1, test material
Material to be tested purchases in Xinjiang Yumin County vibration host safflower development corporation, Ltd., is to adopt current year (2016)
The new seed of receipts, is identified as the seed of compositae plant safflower (Carthamus tinctorius L.), and initial germination percentage is
89.0%, water content 8.1%.Before test, seed is placed in shady and cool ground.
2, test apparatus
It is shown in Table 1
1 test apparatus model of table and producer
3, reagent reagent
Plant soluble sugar kit, BCA method soluble protein kit, superoxide dismutase (SOD) kit,
Peroxidase (POD) kit, (kit is by Suzhou Ke Ming Bioisystech Co., Ltd for catalase (CAT) kit
There is provided), distilled water etc..
4 test methods
4.1 sample preparation
4.1.1 the packaging and storage of seed
Two layers of aluminum foil compound bag heating sealing of Semen Carthami is stored under different temperatures: mid-term library (- 4 ± 2 DEG C),
Long-term library (- 18 ± 2 DEG C) and ultra low temperature freezer (- 75 ± 5 DEG C), take out after freezing January, carry out germination index, conductance to sample
The measurement of rate and physiological and biochemical index.
4.1.2 it thaws when freezing and outbound before storage
According to the progress of plant germplasm resource cryopreservation technology, in conjunction with the actual conditions of germplasm resource bank,
It is as follows to the design scheme of seed storage and outbound:
(1) it is rapidly frozen: directly packaged Semen Carthami being placed under mid-term library and long-term lab environment；It will be loaded on jelly
It deposits the Semen Carthami in pipe and is first placed in liquid nitrogen container and cool down rapidly, then be placed in ultra low temperature freezer.
(2) slowly freezing: packaged seed is subjected to gradient cooling, then is placed under different temperatures.
(3) it quickly thaws: being placed directly in 40 ± 2 DEG C of tepidariums from seed is quickly removed in each temperature library together with packaging,
Defrosting 5min.
(4) it slowly thaws: taking out seed from each temperature library and carry out gradient increased temperature.
The measurement of 4.2 germination indexs
Germination test is carried out referring to the germinating method of " national Seed Inspection regulation " GB/T3543-1995 [7] technical stipulation
Germination test.50 seeds are randomly selected, is placed in the culture dish of two layers of filter paper, is placed in illumination box, germinate item
Part: 25 ± 2 DEG C, 16h/8h.Four repetitions, 2d count germinating energy, and 7d counts germination percentage [8].Each index calculation formula is such as
Under:
Germination percentage=(seed number is planted experimentally in chitting piece grain number/confession) × 100%
Germinating energy=(seed number is planted experimentally in 2d chitting piece grain number/confession) × 100%
Germination index (GI)=Σ (Gt/Dt)
Wherein Gt is the germinative number after t days, and Dt is germination number of days.
The measurement of 4.3 seed leachate conductivity
20, net seed is weighed at random, and weigh W, is rinsed 2 times with deionized water, qualitative filter paper, which blots, swims, and is then placed in
In the conical flask of 100mL, 50mL deionized water is added, measures initial electric conductivity value d1 with DDS-11A type conductivity gauge；20 DEG C of conditions
Lower immersion for 24 hours, measures leachate electric conductivity value d2；Then 35-40min is boiled in boiling water together with the soak of seed, be cooled to 20
After DEG C, deionized water is added to be settled to original volume, the absolute electric conductivity value d3 of rear seed leachate is boiled in measurement.4 repetitions.
Calculation formula is as follows:
Seed leachate conductivity [μ S/ (cmg)]=(d2-d1)/W
Relative conductivity (%)=(d2-d1) × 100/ (d3-d1)
4.4 seed soluble sugars, soluble protein and Antioxidant Enzyme Systems determination of activity
It is carried out according to kit specification operation, measures soluble sugar, soluble protein content and super oxygen in seed
The activity of object mutase (SOD), peroxidase (POD), catalase (CAT), 3 repetitions of each index are averaged.
The analysis of 4.5 data
Data statistics, that is, chart production is carried out using Excel Word 2010.Using 21.0 statistical analysis software of SPSS
ANOVA analysis is carried out to data, is test using Duncan and carries out significance analysis.
5 results and analysis
5.1 freezings and the influence thawed to Semen Carthami germination percentage
In view of the situation of germ plasm resource Kuku temperature, short-term library (15 ± 2 DEG C) Ku Wen and room temperature are close, gradient increased temperature and
It is aobvious that cooling freezes to seed and thaw interrogatory, therefore has studied mid-term library temperature (- 4 ± 2 DEG C), long-term library temperature (- 18 ± 2
DEG C) and ultralow temperature (- 75 ± 5 DEG C) freezing and research of thawing, the variation tendency such as table 2 of germination percentage and Fig. 1 institute after different disposal
Show.As seen from the figure, it have passed through freezing processing before the storage of seeds and without freezing processing, be respectively placed under three kinds of library temperature, after outbound
It has passed through different processing of thawing again, to being placed in Semen Carthami of the short-term library mildly under long-term library temperature, freezing processing and place of thawing
There are few influences on percentage of seedgermination for reason, all remain higher germination percentage level, germination percentage is all 90% or more；It is super to being placed in
Seed under low-temperature condition, the processing of thawing when storage processing being affected for seed, especially outbound, slowly solves out
Jelly is compared to quick-thawing, and germination percentage has dropped 3.23- 5.43%, and the freezing processing before being put in storage then influences seed
Smaller, germination percentage is all >=90%.
The germination percentage (n=4) of each temperature Semen Carthami under the different Techniques of preserving of table 2
Note: reach extremely significant difference (p < 0.01) between colleague's capitalization expression processing, lowercase indicates significant difference
(p<0.05)
5.2 freezings and the influence thawed to Semen Carthami relative conductivity
The relative conductivity variation tendency such as table 3 of Semen Carthami and Fig. 2 institute at each temperature after difference freezing and processing of thawing
Show.It is found that being stored in the Semen Carthami of the mild long-term library temperature in mid-term library, the variation of relative conductivity is without obvious poor between each processing
It is different；And for the Semen Carthami for being stored in ultralow temperature, it freezes and processing of thawing then has a certain impact, before seed storage
Fast freezing and outbound when the relative conductivity under scheme that quickly thaws it is lower.
The relative conductivity (n=4) of Semen Carthami under the different preservation conditions of table 3
Note: reach extremely significant difference (p < 0.01) between colleague's capitalization expression processing, lowercase indicates significant difference
(p<0.05)
5.3 freezings and the influence thawed to Semen Carthami soluble sugar and soluble protein content
Semen Carthami inside storage substance dissolubility sugar and soluble protein contain at each temperature after difference freezing and processing of thawing
Variation tendency is measured as shown in table 4 and Fig. 3.It is found that being stored in the Semen Carthami in mid-term library and long-term library soluble sugar and solvable
No significant difference under the conditions of property protein content is throughout managed；And it is stored in quick solution of the Semen Carthami of ultralow temperature in outbound
Freeze its soluble sugar and soluble protein content is then higher, apparent difference is formed with slow defrosting, at freezing when being put in storage preceding
Reason influences its soluble sugar and soluble protein content little.
The soluble sugar and soluble protein content (n=4) of Semen Carthami under the different preservation conditions of table 4
Note: reach extremely significant difference (p < 0.01) between colleague's capitalization expression processing, lowercase indicates significant difference
(p<0.05)。
5.4 freezings and the influence thawed to Semen Carthami activities of antioxidant enzymes
Semen Carthami activities of antioxidant enzymes variation tendency such as table 5 and Fig. 4 institute at each temperature after difference freezing and processing of thawing
Show.It is found that it is stored in the Semen Carthami in mid-term library and long-term library, the active throughout manage bar of antioxidase (SOD, POD, CAT)
No significant difference under part；And the Semen Carthami for being stored in ultralow temperature then changes significantly between respectively handling.It is stored in mid-term library
Semen Carthami, 4 kinds processing (seed storage before freezing processing and outbound when processing of thawing) under, SOD field of activity exists
Between 52.4-52.8 (U/g fresh weight), under the slow defrosting processing of POD activity and CAT activity in outbound faster at defrosting
The lower height of reason, so slow defrosting when outbound is smaller to Semen Carthami injury.It is stored in the Semen Carthami in long-term library, at 4 kinds
Under reason, antioxidase (SOD, POD, CAT) activity is in p=0.05 and p=0.01 level and no significant difference；SOD activity exists
Lower under the conditions of slow jelly before storage, POD and CAT activity is then slightly higher；Under slow defrosting processing of the CAT activity in outbound slightly
Low height；So the slow jelly alleviation of the Semen Carthami in long-term library is safer.The Semen Carthami being stored under ultralow temperature, 4
Kind processing under, it can be seen that by apparent variation, SOD activity be rapidly frozen and quickly thaw processing under it is lower, POD and
CAT activity is then higher；Quick-thawing when outbound is compared with slow defrosting, and SOD is slightly lower, and POD activity and CAT activity are slightly higher；Institute
It is safer with quick-thawing mode to be rapidly frozen for the Semen Carthami in ultralow temperature.
The activities of antioxidant enzymes (n=4) of Semen Carthami under the different preservation conditions of table 5
Note: reach extremely significant difference (p < 0.01) between colleague's capitalization expression processing, lowercase indicates significant difference
(p<0.05)。
6 discuss
To the Semen Carthami for being stored in mid-term library, the processing of thawing when freezing processing and outbound before seed is put in storage is obtained
On the germination percentage of seed and physio-biochemical characteristics, there are few influences, all remain higher germination percentage level, and germination percentage is held at
92% or more；And physiological and biochemical index all represents under slow thawing condition and is more suitable for seed vitality, and before being put in storage
Freezing processing is then without influence.Thus obtain: Semen Carthami can be placed directly in -4 ± 2 DEG C of mid-term libraries when being put in storage, and when outbound need to be delayed
Slow thaw just can guarantee seed vitality, keep high germination percentage.
It is then to have little impact, the freezing processing before preservation is to percentage of seedgermination to the Semen Carthami for being stored in long-term library
Defrosting processing without influence, but after outbound, germination percentage is then to be declined, and shows as quick-thawing and is less than slowly defrosting；Knot
The variation tendency for closing activities of antioxidant enzymes also show that SOD activity is smaller under slow thaw, and POD activity and CAT activity are slow
It is larger under freezing and slow thawing condition.So the slow jelly alleviation of the Semen Carthami in long-term library is more suitable for.Thus it obtains:
It when Semen Carthami enters -18 ± 2 DEG C of long-term libraries, needs slowly to freeze, germination could both be guaranteed by needing when outbound slowly to thaw
Rate, and guarantee its vigor.
To the seed being placed under ultra low temperature state, each index all shows apparent difference, and storage processing is for seed out
Be affected, especially outbound when processing of thawing, slowly thaw compared to quick-thawing, germination percentage is lower, and is put in storage
Preceding freezing processing then influences seed smaller；Relative conductivity value is higher under slow thawing condition, reserve substance in seed
Content also show as it is higher in quick-frozen fast solution condition；In conjunction with the variation tendency of activities of antioxidant enzymes, obtain in ultralow temperature
Semen Carthami for, be rapidly frozen and quick-thawing mode it is safer.It may be because defrosting thaws speed by this temperature
Area, to avoid intracellular secondary icing.Thus it obtains: when Semen Carthami enters -75 ± 2 DEG C of ultralow cold storages, needing quickly
Freezing, when outbound, need quick-thawing, could not only guarantee percentage of seedgermination, but also guarantee its vigor.
To sum up, the Semen Carthami of cryopreservation through the invention goes out to be put in storage freeze-thaw method, obtained safflower kind
Sub- germination percentage is high, and SOD activity and relative conductivity are lower, and POD and CAT activity is higher, the content of reserve substance in seed
Height has application value.
Claims (8)
1. a kind of Semen Carthami of cryopreservation goes out storage method, it is characterised in that:
1) when being stored in mid-term library: Semen Carthami is placed directly within the storage of -4 ± 2 DEG C of seed banks, and when outbound, gradient increased temperature is to normal
Temperature；
2) when being stored in long-term library: Semen Carthami is first stood at 2~8 DEG C, then is stored in -18 ± 2 DEG C of seed banks, when outbound,
Gradient increased temperature is to room temperature；
3) when being stored in ultralow cold storage: Semen Carthami is first placed in liquid nitrogen and freezes, then is stored in -75 ± 5 DEG C of seed banks, outbound
When, it is first placed in tepidarium and thaws, then be placed at room temperature for；
The gradient increased temperature to room temperature is first 2~8 DEG C of standings, then tepidarium, is finally placed at room temperature for.
2. freeze-thaw method according to claim 1, it is characterised in that: step 1) and the step 2) Semen Carthami are
Sealed package is heated with two layers of aluminum foil compound bag.
3. going out storage method according to claim 1, it is characterised in that: 4 DEG C of the step 2) dwell temperature, the time 24~
30h, preferably for 24 hours.
4. going out storage method according to claim 1, it is characterised in that: the step 3) Semen Carthami is loaded on cryopreservation tube
In.
5. going out storage method according to claim 1, it is characterised in that: step 3) is described to be placed in 5~10min of liquid nitrogen time,
It is preferred that 5min.
6. going out storage method according to claim 1, it is characterised in that: the temperature of the tepidarium is 40 ± 2 DEG C, the time 5
~10min, preferably 5min.
7. going out storage method according to claim 1, it is characterised in that: the gradient increased temperature to room temperature is first to stand for 2~8 DEG C
5~10min, then 40 ± 2 DEG C of 5~10min of water-bath, are finally placed at room temperature for.
8. going out storage method according to claim 7, it is characterised in that: the gradient increased temperature to room temperature is first to stand for 4 DEG C
5min, then 40 ± 2 DEG C of water-bath 5min, are finally placed at room temperature for.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114568135A (en) * | 2022-03-25 | 2022-06-03 | 成都中医药大学 | Preservation method and germination method of evergreen fruit tree seeds |
| CN117315490A (en) * | 2023-11-23 | 2023-12-29 | 成都中医药大学 | Method for detecting germination rate of seeds, model, storage medium and modeling method |
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| CN1695415A (en) * | 2004-05-10 | 2005-11-16 | 新昌县苦丁茶研究所 | Method for storing seeds of sassafras |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114568135A (en) * | 2022-03-25 | 2022-06-03 | 成都中医药大学 | Preservation method and germination method of evergreen fruit tree seeds |
| CN114568135B (en) * | 2022-03-25 | 2023-10-20 | 成都中医药大学 | Preservation method and germination method of evergreen fruit tree seeds |
| CN117315490A (en) * | 2023-11-23 | 2023-12-29 | 成都中医药大学 | Method for detecting germination rate of seeds, model, storage medium and modeling method |
| CN117315490B (en) * | 2023-11-23 | 2024-02-02 | 成都中医药大学 | Method for detecting germination rate of seeds, model, storage medium and modeling method |
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