CN104255706A - Method for optimizing vitrification ultra-low temperature preservation effect of arabidopsis seedlings - Google Patents
Method for optimizing vitrification ultra-low temperature preservation effect of arabidopsis seedlings Download PDFInfo
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Abstract
The invention discloses a method for optimizing a vitrification ultra-low temperature preservation effect of arabidopsis seedlings. According to the invention, the arabidopsis seedlings are processed by adopting a vitrification solution which contains a carbon nanometer material so as to improve the preservation effect of the arabidopsis seedlings. The method specifically comprises the steps of loading liquid treatment, vitrification solution treatment and liquid nitrogen preservation, wherein the vitrification solution is the MS culture solution containing 0.1-0.5 g/L of grapheme quantum dots. The method disclosed by the invention optimizes the preservation effect of the arabidopsis seedlings remarkably; as the grapheme quantum dots are added to serve as an allogenic material, the vitrification ultra-low temperature preservation of plants is facilitated.
Description
Technical field
The present invention relates to the preservation field of plant or its local, be specifically related to a kind of method optimizing Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect.
Background technology
Excised Embryos is the modern germ plasm resource Plantlet in vitro technology grown up the seventies in last century.Usually preserve in liquid nitrogen, be saved metabolism in Materials Cell and vegetative activity almost stops completely, be in metastable biological condition, reach the object of long-term conserving species matter, Excised Embryos is the medium-term and long-term preserving type uniquely not needing continuous subculture at present.Cryopreservation by vitrification cell or tissue is placed in the vitrification solution be made up of a certain proportion of permeability and impermeability protectant; make material and vitrification solution thereof under enough fast rate of temperature fall, be solidified into amorphous glassy state, and preserve at low temperatures with this glassy state.Vitrification is because of simple and quick, and cost is low, is suitable for preserving kind extensively, and preserve material genetic stability, the advantages such as preservation effect is good are nearly ten years for the prefered method of the medium-term and long-term preservation of fine germplasm resources.
In the method that the vitrification ultra-low temperature of plant and tissue thereof is preserved, when adopting some store methods, plant and organized renewing thereof cultivate after survival rate not high or can not survive at all; When adopting identical store method in addition, whether different plants and organized renewing thereof can survive after cultivating and the height of survival rate is also not quite similar.Add certain allogenic material in prior art to be conducive to improving Excised Embryos survival rate, but it is but unknown for could improving its preservation effect for specific plant and which kind of allogenic material of organizational choice.
Arabidopsis is in Angiospermae, and Dicotyledoneae, it is 2 years sward bases, and arabidopsis is that plant is little, knot is many as the advantage of model plant, and it is also self-pollination plant, gene high homogenous.In the research in Excised Embryos field, arabidopsis has consequence and Research Significance.Arabidopsis thaliana Seedlings is extraordinary research material, find in existing research, arabidopsis sprouting 60h seedling can be screened different additives and verify in the impact of Excised Embryos effect, can, as the molecule mechanism of preserving in investigation of materials Cryopreservation, be a kind of very important physiology and molecular biology research material simultaneously.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide a kind of method optimizing Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect, to overcome the shortcoming that in prior art, Arabidopsis thaliana Seedlings Excised Embryos recovery percentage is low, and optimize the Physiology and biochemistry response investigations of plant vitrification ultra-low temperature preservation.
To achieve these goals or other objects, the present invention is achieved by the following technical solutions.
Optimize a method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect, the method adopting vitrification ultra-low temperature to preserve is preserved Arabidopsis thaliana Seedlings, and concrete steps are as follows:
1) load liquid process: under room temperature, Arabidopsis thaliana Seedlings immersion treatment in loading liquid was removed loading liquid after 20 ~ 30 minutes;
2) vitrification solution process: use vitrification solution immersion treatment Arabidopsis thaliana Seedlings 40 ~ 60 minutes at 0 ~ 25 DEG C;
3) Liquid nitrogen storage: keep the Arabidopsis thaliana Seedlings state of soaking in vitrification solution, and be placed in liquid nitrogen and preserve;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot.
The culture fluid of MS described in the present invention contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/L KH
2pO
4, 370mg/L MgSO
47H
2o, 440mg/L CaCl
22H
2o, 37.3mg/L Na
2-EDTA, 27.8mg/L FeSO
47H
2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/L ZnSO
47H
2o, 0.25mg/L Na
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/L CoCl
26H
2o, surplus is water.The pH of described MS culture fluid is 5.8.
Preferably, described Arabidopsis thaliana Seedlings is the Arabidopsis thaliana Seedlings after cultivating on MS solid culture medium, and the method that MS medium is cultivated is: be placed in by arabidopsis seed on MS solid culture medium, every day first carries out illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m
2, temperature 20 ~ 27 DEG C, every day, illumination cultivation carried out dark culturing remaining time, and dark culturing temperature is 20 ~ 25 DEG C, circulation illumination cultivation every day and dark culturing.
The solid culture medium of MS described in the present invention contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/L KH
2pO
4, 370mg/L MgSO
47H
2o, 440mg/L CaCl
22H
2o, 37.3mg/L Na
2-EDTA, 27.8mg/LFeSO
47H
2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/L ZnSO
47H
2o, 0.25mg/L Na
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/L CoCl
26H
2o, 30g/L sucrose, 10g/L agar powder, surplus is water.The pH of described MS solid culture medium is 5.8.
Preferably, the time that described arabidopsis seed is cultivated on pre-culture medium is 24 ~ 72h.
More preferably, the time that described arabidopsis seed is cultivated on pre-culture medium is 60h.
The method that the seed of arabidopsis described in the present invention is cultivated on MS medium be adopt every day illumination cultivation and dark culturing to replace form until reach 60h.As, first carry out the illumination cultivation of 8h every day, then carry out the dark culturing of 16h, so circulation is until the 3rd day cultivates 12h again, and the 12h of the 3rd day is illumination cultivation.
Preferably, under room temperature, Arabidopsis thaliana Seedlings immersion treatment in loading liquid was removed loading liquid after 20 minutes.
Preferably, at 0 DEG C, vitrification solution immersion treatment Arabidopsis thaliana Seedlings 50 minutes are used.
More preferably, the method that described Arabidopsis thaliana Seedlings is cultivated on MS medium is: first carry out 8h illumination cultivation, light intensity 100 ~ 200 μm of ol/m
2, temperature 25 DEG C; Carry out 16h dark culturing again, dark culturing temperature is 20 DEG C.More preferably, light intensity is 150 μm of ol/m
2.
Preferably, described arabidopsis seed is the arabidopsis seed first crossed through vernalization, and described vernalization is that described arabidopsis seed is placed 24 ~ 72h at 0 ~ 10 DEG C.
Preferably, described vernalization is that described arabidopsis seed is placed 40 ~ 50h at 4 ~ 6 DEG C.
More preferably, described vernalization for place 48h by described arabidopsis seed at 4 DEG C.
Preferably, described arabidopsis seed is first sterile-processed before vernalization, disinfecting technique is: by arabidopsis seed 65 ~ 75% alcohol immersion 10 ~ 30s, re-use aseptic water washing, then use the aqueous solution soaking 5 ~ 15 minutes containing the NaClO of 15 ~ 25wt% and the polysorbas20 of 0 ~ 0.03wt%, and use aseptic water washing.65 ~ 75% ethanol described in the present invention refer to the aqueous solution of ethanol, and wherein percentage composition is volumn concentration.
More preferably, described technique of disinfecting is: by arabidopsis seed 70% ethanol disinfection 15s, aseptic water washing 4 ~ 6 times, then uses the aqueous solution soaking 10 minutes containing the NaClO of 15 ~ 25wt% and the polysorbas20 of 0 ~ 0.03wt%, and uses aseptic water washing 5 ~ 6 times.
More preferably, disinfect technique and be described in: by arabidopsis seed 70% ethanol disinfection 15s, aseptic water washing 4 times, then uses the aqueous solution soaking 10 minutes of the polysorbas20 of NaClO and 0.01wt% containing 20wt%, and use aseptic water washing 6 times.
Preferably, described loading liquid is the MS culture fluid containing 1 ~ 2mol/L glycerine and 0.3 ~ 0.5mol/L sucrose.
Preferably, described arabidopsis seed is Columbia ecotype arabidopsis seed as Arabidopsis thaliana ecotype Col-0 type.
Preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
More preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.3g/L graphene quantum dot.
More preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1g/L graphene quantum dot, or described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.3g/L graphene quantum dot.
The invention also discloses thawing and cultural method again of a kind of Arabidopsis thaliana Seedlings, described Arabidopsis thaliana Seedlings is the Arabidopsis thaliana Seedlings adopting method described above to preserve, described Arabidopsis thaliana Seedlings thaw and again cultural method for Arabidopsis thaliana Seedlings is taken out from liquid nitrogen, first water-bath is thawed, then wash with cleaning solution after removing vitrification solution, finally proceed to renewal cultivation in MS solid culture medium.
Preferably, the condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C, by the technique of cleaning solution washing be: at room temperature soak 30 ~ 50 minutes with cleaning solution, and changing once washing liquid every 5 ~ 10 minutes, described cleaning solution is the MS culture fluid containing 1.0 ~ 1.5mol/L sucrose.
More preferably, the condition that water-bath is thawed is the 90s that thaws in the water-bath of 40 DEG C, by the technique of cleaning solution washing be: at room temperature soak 40 minutes with cleaning solution, and changed once washing liquid every 10 minutes, described cleaning solution is the MS culture fluid containing 1.2mol/L sucrose.
More preferably, shake acceleration is adopted to thaw scheme when water-bath is thawed.
Preferably, described cultural method is again by Arabidopsis thaliana Seedlings every day first illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m
2, cultivation temperature is 20 ~ 27 DEG C, and in every day, remaining time adopts dark culturing, and dark culturing temperature is 20 ~ 25 DEG C.
More preferably, described renewal cultivation technique is by Arabidopsis thaliana Seedlings every day first illumination cultivation 8h, light intensity 150 μm of ol/m
2, cultivation temperature is 25 DEG C, and in every day, remaining time adopts dark culturing, and dark culturing temperature is 20 DEG C
More preferably, in order to prove the preservation effect of method in the present invention, recovery percentage is adopted to add up, particularly, in the quantity of adding up the Arabidopsis thaliana Seedlings survived on the 15th day of renewal cultivation, and calculating recovery percentage, described recovery percentage is: the seedling numbers survived/seedling total quantity × 100%.
According to nano science principle, in cryoprotector, add viscosity and thermal conductivity that nano material effectively can improve cryoprotector, change the formation situation of ice crystal, reduce injury to cell.The present invention is to Arabidopsis thaliana Seedlings, particularly Seed Germination of Arabidopsis Pumila 60h seedling is in cryopreservation by vitrification, first with loading liquid process, then vitrification solution process is adopted, finally Excised Embryos in liquid nitrogen, wherein vitrification solution is again for the addition of the vitrification solution of graphene quantum dot, and other techniques in the interpolation fitting method of this allogenic material effectively can improve the preservation effect of Arabidopsis thaliana Seedlings.Method disclosed in the present invention is remarkable to the preservation effect optimization of Arabidopsis thaliana Seedlings, plays facilitation by adding graphene quantum dot as allogenic material to the preservation of plant vitrification ultra-low temperature.
Accompanying drawing explanation
Fig. 1 is 60h Arabidopsis thaliana Seedlings Excised Embryos restoration ecosystem photo in experimental group and control group.
In Fig. 1, left column is 60h Arabidopsis thaliana Seedlings Excised Embryos restoration ecosystem photo in control group;
In Fig. 1, the right side is classified as 60h Arabidopsis thaliana Seedlings Excised Embryos restoration ecosystem photo in experimental group;
In Fig. 1, the first content being discharged to the graphene quantum dot added in vitrification solution in the 3rd row is respectively 0.1g/L, 0.3g/L and 0.5g/L.
Embodiment
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this specification also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Arabidopsis seed used in the embodiment of the present invention is arabidopsis Columbia ecotype seed, Arabidopsis thaliana ecotype Col-0 type.
In the embodiment of the present invention, the formula of experiment reagent is as follows:
1) MS culture fluid is: MS culture fluid contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/L KH
2pO
4, 370mg/L MgSO
47H
2o, 440mg/L CaCl
22H
2o, 37.3mg/L Na
2-EDTA, 27.8mg/L FeSO
47H
2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/L ZnSO
47H
2o, 0.25mg/L Na
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/L CoCl
26H
2o, surplus is water, and the pH of described MS culture fluid is 5.8.
2) MS solid culture medium is: MS solid culture medium contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/L KH
2pO
4, 370mg/L MgSO
47H
2o, 440mg/L CaCl
22H
2o, 37.3mg/L Na
2-EDTA, 27.8mg/L FeSO
47H
2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/L ZnSO
47H
2o, 0.25mg/L Na
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/L CoCl
26H
2o, 30g/L sucrose, 10g/L agar powder, surplus is water, and the pH of described MS solid culture medium is 5.8.
3) loading liquid is: the MS culture fluid containing 2mol/L glycerine and 0.4mol/L sucrose.
4) vitrification solution is: containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
5) cleaning solution is the MS culture fluid containing 1.2mol/L sucrose.
Embodiment 1
1) acquisition of Arabidopsis thaliana Seedlings: by arabidopsis seed 70% ethanol disinfection 15s, again with the aqueous solution sterilization 10min containing 20wt%NaClO and 0.01wt% polysorbas20 after aseptic water washing 4 times, aseptic water washing 6 times; Then by sterilization after seed at 4 DEG C of vernalization 48h, be placed on MS solid culture medium and cultivate, condition of culture is illumination cultivation 8h every day, light intensity 150 μm of ol/m
2, cultivation temperature 25 DEG C; Then dark culturing 16h, cultivation temperature is 20 DEG C, altogether by seed culture 60h, obtains the Arabidopsis thaliana Seedlings of 60h seedling age.
2) 60h Arabidopsis thaliana Seedlings is divided into experimental group and control group, establishes 3 parallel laboratory tests respectively for each group, 50 strain seedling are put into the 2mL cryovial that 1mL loads liquid is housed, room temperature treatment 20min; Liquid will be loaded absorb, add vitrification solution, 0 DEG C of process 50min; Cryovial is dropped in liquid nitrogen and preserves 1h.
Graphene quantum dot respectively containing 0.1g/L, 0.3g/L, 0.5g/L in the vitrification solution of experimental group.
Graphene quantum dot containing 0.1g/L in the vitrification solution of experimental group group 1; Not containing graphene quantum dot in control group group 1, other are identical with experimental group group 1;
Graphene quantum dot containing 0.3g/L in the vitrification solution of experimental group group 2; Not containing graphene quantum dot in control group group 2, other are identical with experimental group group 2;
Graphene quantum dot containing 0.5g/L in the vitrification solution of experimental group group 3; Not containing graphene quantum dot in control group group 3, other are identical with experimental group group 3.
3), after cryovial preserves 1h in liquid nitrogen, take out cryovial, put into 40 DEG C of water-baths fast, thaw 90s, and frequently shake gently; Vitrification solution is absorbed, adds cleaning solution, room temperature treatment 40min, change once washing liquid every 10min; Seedling after washing moves on in MS solid culture medium, puts into plant incubator, the same Germination Condition of incubator parameters, namely every day illumination cultivation 8h, light intensity 150 μm of ol/m
2, cultivation temperature 25 DEG C; Then dark culturing 16h, cultivation temperature is 20 DEG C; Arabidopsis thaliana Seedlings renewal cultivation the 15th day, calculates and the recovery percentage of 60h Arabidopsis thaliana Seedlings in comparative experiments group and control group.
The recovery percentage of experimental group and control group Arabidopsis thaliana Seedlings is in table 1.
Table 1
3. experimental result
As shown in Table 1, be that the graphene quantum dot of 0.1g/L, 0.3g/L, 0.5g/L adds in the vitrification solution sprouting 60h Arabidopsis thaliana Seedlings Excised Embryos by concentration, 60h Arabidopsis thaliana Seedlings recovery percentage is made to bring up to 38.97%, 46.80% and 41.52% by 25.54% respectively, the improvement effect taking concentration as the graphene quantum dot of 0.3g/L is the most remarkable, specifically sees Fig. 1.
Embodiment 2
The present embodiment mainly carries out test analysis to Arabidopsis thaliana Seedlings in the parameter in each stage of Excised Embryos, thus proves the regulating and controlling effect of external source graphene quantum dot to membrane damage and oxidative stress in arabidopsis 60h seedling Cryopreservation.
1) experiment material: in group 1, material is: arabidopsis sprouts the seedling of 60h according to embodiment in the present invention 1 record method
In group 2, material is: the seedling in embodiment 1 after vitrification solution process;
Group 3 is: the seedling after thawing after the preservation of method vitrification ultra-low temperature in embodiment 1;
Group 4 be in embodiment 1 through cleaning solution wash after seedling;
Group 5 is the seedling in embodiment 1 after renewal cultivation process.
1) experiment material: arabidopsis sprouts that committed step in 60h seedling and Cryopreservation is dewatered, thaws, washed, seedling after renewal cultivation process.
2) experimental technique:
1, the mensuration of relative conductivity in arabidopsis 60h seedling Cryopreservation
Get the Arabidopsis thaliana Seedlings after above-mentioned 5 phase process, repeat for 3 times.Rinse for several times with double distilled water, then blot surface moisture with filter paper, load 50mL beaker, add 50mL double distilled water and soak 2h at 25 DEG C, survey its conductance R with Thermo Orion FE30 type conductivity gauge.Survey and finish, by each beaker plastic film sealing, be placed in take out after boiling water bath boils 15min and be cooled to 25 DEG C, shake up, survey electric conductivity value R
0, according to following formulae discovery relative conductivity:
2, in arabidopsis 60h seedling Cryopreservation, MDA measures
Take the mortar that precooling put into by 0.2g material, add 5mL 10% trichloroacetic acid (TCA) and be ground to homogenate, pour the centrifugal 10min of 5000rpm in 10mL centrifuge tube into, supernatant is MDA liquid to be measured.Aspirate supernatant 2mL adds 2mL 0.6% thiobarbituricacidα-(TBA) solution, reacts 30min after mixing in boiling water bath, rapidly the centrifugal 10min of 5000rpm after cooling, colorimetric estimation respectively under 600nm, 532nm, 450nm wavelength.Return to zero with 2mL distilled water.
MDA concentration C in extract (
μmoL/L)=6.45 (OD
532– OD
600) – 0.56OD
450, the content being converted into MDA in every gram of material is:
3, the mensuration of content of hydrogen peroxide in arabidopsis 60h seedling Cryopreservation
Build up content of hydrogen peroxide according to Nanjing and measure kit specification mensuration.
3) experimental result
Relative conductivity, malonaldehyde and content of hydrogen peroxide is utilized to have studied the regulating and controlling effect of external source graphene quantum dot to cell membrane damage, Lipid peroxidation metabolism and the oxidative stress in 60h Arabidopsis thaliana Seedlings Cryopreservation.Experimental result shows, and significantly reduces the cell membrane damage degree in Cryopreservation, effectively reduce content of hydrogen peroxide, thus reduce Lipid peroxidation metabolism degree after adding 0.3g/L graphene quantum dot material.0.3g/L graphene quantum dot has been confirmed to the protective effect 60h Arabidopsis thaliana Seedlings Cryopreservation from Physiology and biochemistry and cytology angle.Concrete outcome is in table 2, table 3 and table 4.Wherein in table 2, table 3 and table 4, control group is control group in embodiment 1, and experimental group is the experimental group of adding 0.3g/L graphene quantum dot in embodiment 1.
The change of table 2 control group and experimental group relative conductivity
| Relative conductivity (%) | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 |
| Control group | 29.78 | 80.09 | 86.78 | 84.23 | 83.72 |
| Experimental group | 29.78 | 74.32 | 79.89 | 77.14 | 76.56 |
The change of table 3 control group and experimental group mda content
The change of table 4 control group and experimental group content of hydrogen peroxide
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (10)
1. optimize a method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect, it is characterized in that, the method adopting vitrification ultra-low temperature to preserve is preserved Arabidopsis thaliana Seedlings, and concrete steps are as follows:
1) load liquid process: under room temperature, Arabidopsis thaliana Seedlings immersion treatment in loading liquid was removed loading liquid after 20 ~ 30 minutes;
2) vitrification solution process: use vitrification solution immersion treatment Arabidopsis thaliana Seedlings 40 ~ 60 minutes at 0 ~ 25 DEG C;
3) Liquid nitrogen storage: keep Arabidopsis thaliana Seedlings to be immersed in state in vitrification solution, and be placed in liquid nitrogen and preserve;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot.
2. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described Arabidopsis thaliana Seedlings is the Arabidopsis thaliana Seedlings after cultivating on MS solid culture medium, the method that MS solid culture medium is cultivated is: be placed in by arabidopsis seed on MS solid culture medium, every day first carries out illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m
2, temperature 20 ~ 27 DEG C, every day, illumination cultivation carried out dark culturing remaining time, and dark culturing temperature is 20 ~ 25 DEG C, circulation illumination cultivation every day and dark culturing.
3. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 2, it is characterized in that, described arabidopsis seed is the arabidopsis seed first crossed through vernalization, and described vernalization is that described arabidopsis seed is placed 24 ~ 72h at 0 ~ 10 DEG C.
4. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 3, it is characterized in that, described vernalization is that described arabidopsis seed is placed 40 ~ 50h at 4 ~ 6 DEG C.
5. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 3, it is characterized in that, described arabidopsis seed is first sterile-processed before vernalization, disinfecting technique is: by arabidopsis seed 65 ~ 75% alcohol immersion 10 ~ 30s, re-use aseptic water washing, then use the aqueous solution soaking 5 ~ 15 minutes containing the NaClO of 15 ~ 25wt% and the polysorbas20 of 0 ~ 0.03wt%, and use aseptic water washing.
6. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described loading liquid is the MS culture fluid containing 1 ~ 2mol/L glycerine and 0.3 ~ 0.5mol/L sucrose.
7. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
8. the thawing and cultural method again an of Arabidopsis thaliana Seedlings, described Arabidopsis thaliana Seedlings is for adopting the Arabidopsis thaliana Seedlings that as described in claim as arbitrary in claim 1 ~ 7, method is preserved, described Arabidopsis thaliana Seedlings thaw and again cultural method for Arabidopsis thaliana Seedlings is taken out from liquid nitrogen, first water-bath is thawed, then wash with cleaning solution after removing vitrification solution, finally proceed to renewal cultivation in MS solid culture medium.
9. thaw as claimed in claim 8 and cultural method again, it is characterized in that, the condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C, by the technique of cleaning solution washing be: at room temperature soak 30 ~ 50 minutes with cleaning solution, and changing once washing liquid every 5 ~ 10 minutes, described cleaning solution is the MS culture fluid containing 1.0 ~ 1.5mol/L sucrose.
10. thaw as claimed in claim 8 and cultural method again, it is characterized in that, described cultural method is again by Arabidopsis thaliana Seedlings every day first illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m
2, cultivation temperature is 20 ~ 27 DEG C, and in every day, remaining time adopts dark culturing, and dark culturing temperature is 20 ~ 25 DEG C.
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| CN106256196A (en) * | 2015-11-20 | 2016-12-28 | 沈阳善达生物科技有限公司 | Efficiently induce method and the inducing culture of Colombia's type Arabidopsis callus |
| CN106613838A (en) * | 2016-11-29 | 2017-05-10 | 中国科学院昆明植物研究所 | Method for increasing regeneration rate after Arabidopsis thaliana stem tips are preserved at ultra-low temperature |
| CN109319770A (en) * | 2018-09-12 | 2019-02-12 | 东莞理工学院 | Solution acid alkalinity adjusting method based on graphene quantum dot |
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| CN110959330A (en) * | 2019-11-13 | 2020-04-07 | 上海交通大学 | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104604539A (en) * | 2015-01-30 | 2015-05-13 | 山东省林业科学研究院 | Grafting method for arabidopsis |
| CN106256196A (en) * | 2015-11-20 | 2016-12-28 | 沈阳善达生物科技有限公司 | Efficiently induce method and the inducing culture of Colombia's type Arabidopsis callus |
| CN106256196B (en) * | 2015-11-20 | 2018-05-29 | 刘寒冬 | The efficiently method and inducing culture of induction Colombia type Arabidopsis callus |
| CN106613838A (en) * | 2016-11-29 | 2017-05-10 | 中国科学院昆明植物研究所 | Method for increasing regeneration rate after Arabidopsis thaliana stem tips are preserved at ultra-low temperature |
| CN109319770A (en) * | 2018-09-12 | 2019-02-12 | 东莞理工学院 | Solution acid alkalinity adjusting method based on graphene quantum dot |
| CN109452265A (en) * | 2018-11-13 | 2019-03-12 | 上海交通大学 | It reduces Cellular stress injury and improves the Y of cryopreservation effect2SK2Dehydrins |
| CN109619094A (en) * | 2018-12-13 | 2019-04-16 | 上海交通大学 | Afriocan agapanthus SK3Dehydrin protein is reducing Cellular stress injury and is improving the application in cryopreservation effect |
| CN109619094B (en) * | 2018-12-13 | 2021-05-28 | 上海交通大学 | Baizilian SK3Application of dehydrin protein in reducing cell stress injury and improving ultralow temperature preservation effect |
| CN110301434A (en) * | 2019-07-15 | 2019-10-08 | 中国农业科学院作物科学研究所 | A kind of rice seedling cryopreservation method |
| CN110959330A (en) * | 2019-11-13 | 2020-04-07 | 上海交通大学 | Application of agapanthus cystatin in improving survival rate of plant cells after ultralow-temperature preservation |
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