CN104170818A - Method for optimizing vitrified cryopreservation effect of agapanthus embryonic calluses - Google Patents
Method for optimizing vitrified cryopreservation effect of agapanthus embryonic calluses Download PDFInfo
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- CN104170818A CN104170818A CN201410468264.XA CN201410468264A CN104170818A CN 104170818 A CN104170818 A CN 104170818A CN 201410468264 A CN201410468264 A CN 201410468264A CN 104170818 A CN104170818 A CN 104170818A
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- embryo callus
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Abstract
The invention discloses a method for optimizing a vitrified cryopreservation effect of agapanthus embryonic calluses. Cymbidium protocorms are processed by a vitrification solution containing carbon nanomaterials to improve the preservation effect. The method specifically comprises the following steps: preculturing; processing of a loading solution; processing of the vitrification solution; and preserving liquid nitrogen, wherein the vitrification solution contains 0.1-0.5g/L graphene quantum dots. According to the method disclosed by the invention, the preservation effect on the agapanthus embryonic calluses is significantly optimized, and the graphene quantum dots are added as an allogenic material to accelerate cryopreservation of plants by vitrification.
Description
Technical field
The present invention relates to the preservation field of plant or its part, be specifically related to a kind of method of optimizing Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect.
Background technology
It is the in vitro Techniques of preserving of modern germ plasm resource growing up the seventies in last century that ultralow temperature is preserved.Conventionally in liquid nitrogen, preserve, being saved the intracellular metabolism of material and vegetative activity almost stops completely, be in metastable biological condition, reach the object of long-term preservation germplasm, it is current unique medium-term and long-term preserving type that does not need continuous subculture that ultralow temperature is preserved.Cryopreservation by vitrification is that cell or tissue is placed in to the vitrification solution being comprised 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.The advantages such as Vitrification is because of simple and quick, and cost is low, and suitable preservation kind is extensive, preserves material genetic stability, and preservation effect is good are nearly ten years for the prefered method of the medium-term and long-term preservation of fine germplasm resources.
Afriocan agapanthus is called blue lily, Afric lilium, is Agapanthaceae Agapanthus perennial plant, originates in south, Africa.Because of its plant tall and straight, leaf beautiful, leaf look dark green, flower amount is large, bright in luster and enjoy liking of people, has become abroad common ornamental flower, is used for garden cultivation and cut-flower production; In addition, Afriocan agapanthus also has that to fix the sand the Ecology Action of bank protection and analgesia etc. medicinal.China introduces the time of Afriocan agapanthus and does not grow (2000), and the ripening rate in Shanghai is low, depend on external import seminal propagation, therefore, the preservation of the Vitro Quick Reproduction occurring by somatic embryo and Afriocan agapanthus germ plasm resource is most important to the demand of Afriocan agapanthus high quality seedling more.Correlative study has been set up Afriocan agapanthus embryo callus ultralow temperature and has been preserved system, and after preserving, the relative survival rate of cell has reached 56.94%, but still is not enough to meet the demand of production and scientific research.
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 of new optimization Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, to overcome in prior art plant and to organize medium-term and long-term preservation to be difficult to realize, and ultralow temperature plant or the low shortcoming of its organized renewing growth rate of preserving.
To achieve these goals or other objects, the present invention is achieved by the following technical solutions.
Optimize a method for Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, the method that adopts vitrification ultra-low temperature to preserve is preserved Afriocan agapanthus embryo callus, and concrete steps are as follows:
1) preculture: Afriocan agapanthus embryo callus is placed on pre-culture medium, and preculture is 1～4 day at 0～10 ℃;
2) loading liquid processes: under room temperature, Afriocan agapanthus embryo callus immersion treatment in loading liquid is removed to loading liquid after 40～60 minutes;
3) vitrification solution is processed: at 0～25 ℃, use vitrification solution to soak dehydration processing Afriocan agapanthus embryo callus 40～60 minutes;
4) liquid nitrogen is preserved: the state that keeps Afriocan agapanthus embryo callus to be soaked by vitrification solution, and be placed in liquid nitrogen
Preserve;
Described vitrification solution is the glass freezing protection liquid that contains 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 pre-culture medium is the MS solid culture medium that contains 0.4～0.8mol/L sucrose.
Preferably, described pre-culture medium is the MS solid culture medium that contains 0.5mol/L sucrose.
Preferably, described 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, the pH of described MS solid culture medium is 5.8.
Preferably, above-mentioned steps 1) described in Afriocan agapanthus embryo callus for the method for cultivating on pre-culture medium be: Afriocan agapanthus embryo callus is placed at 4 ℃ on pre-culture medium 1～4 day.
Preferably, above-mentioned steps 1) described in Afriocan agapanthus embryo callus for the method for cultivating on pre-culture medium be: Afriocan agapanthus embryo callus is placed at 4 ℃ to pre-culture medium upper 2 day.
Preferably, described loading liquid is for containing 1～2mol/L glycerine, 0.3～0.5mol/L sucrose and 5～10mmol/L KNO
3mS culture fluid.
Preferably, described loading liquid is for containing 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
Preferably, above-mentioned steps 2) in, under room temperature, Afriocan agapanthus embryo callus immersion treatment in loading liquid is removed to loading liquid after 60 minutes;
Preferably, above-mentioned steps 3) in, at 0 ℃, use vitrification solution to soak dehydration processing Afriocan agapanthus embryo callus 40 minutes.
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.
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.
Thawing and cultural method again of a kind of Afriocan agapanthus embryo callus, the Afriocan agapanthus embryo callus of described Afriocan agapanthus embryo callus for adopting method as described above to preserve, described Afriocan agapanthus embryo callus thaw and again cultural method for Afriocan agapanthus embryo callus is taken out from liquid nitrogen, first water-bath is thawed, then after removing vitrification solution, with cleaning solution, wash, finally proceed to renewal cultivation in recovery media.
Preferably, the condition that water-bath is thawed for thawing 60～120s in the water-bath of 30～40 ℃.
More preferably, the condition that water-bath is thawed for thawing 90s in the water-bath of 40 ℃.
When more preferably, water-bath is thawed, adopt to shake and accelerate to thaw scheme.
Preferably, described cleaning solution is for containing 1.0～1.5mol/L sucrose and 5～10mmol/L KNO
3mS culture fluid.
Preferably, described cleaning solution is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS culture fluid, washing process is: with cleaning solution, at room temperature Afriocan agapanthus embryo callus is soaked 10～30 minutes.
More preferably, described washing process is: with cleaning solution, at room temperature process 30 minutes, change once washing liquid every 10 minutes.
Preferably, described recovery media is the MS medium that contains 1.0～3.0mg/L picloram and 30g/L sucrose.
Preferably, described recovery media is the MS medium that contains 1.5mg/L picloram and 30g/L sucrose.
In the present invention, disclosed method is applicable to all Afriocan agapanthus embryo callus in prior art.Preferably, preparation method's list of references of described Afriocan agapanthus embryo callus: the research of blue Rapid Propagation of Lilium technology, Fan Xianli, Shanghai Communications University's master thesis, 2009.
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 0.3g/L graphene quantum dot.
More preferably, adopt relative survival to add up, particularly, at renewal cultivation, after 30 days, utilize TTC (being abbreviated as TTC) legally constituted authority meter Afriocan agapanthus embryo callus relative survival, and be aided with the observation of fluorescein(e) diacetate (being abbreviated as FDA) decoration method.
According to nano science principle, in cryoprotector, add viscosity and the thermal conductivity that nano material can effectively improve cryoprotector, change formation situation, the injury of minimizing to cell of ice crystal.The present invention preserves under vitrification ultra-low temperature condition Afriocan agapanthus embryo callus, first carry out preculture, then successively with loading liquid, vitrification solution processing, wherein vitrification solution is again for having added the vitrification solution of graphene quantum dot, other techniques in the interpolation fitting method of this allogenic material, can effectively improve the preservation effect of Afriocan agapanthus embryo callus.According to store method disclosed by the invention, when the graphene quantum dot that employing concentration is 0.1～0.5g/L is used as allogenic material, recovery percentage after Afriocan agapanthus embryo callus vitrification ultra-low temperature is preserved significantly improves, in the present invention, disclosed method is remarkable to the preservation effect optimization of Afriocan agapanthus embryo callus, by adding graphene quantum dot, as allogenic material, plant vitrification ultra-low temperature is preserved and is played facilitation.
Accompanying drawing explanation
Fig. 1 is the FDA dyeing photo of Afriocan agapanthus embryo callus ultralow temperature saving/restoring growth in experimental group and control group.FDA can enter in living cells protoplast and produce fluorescence, can be used as judgement cell sign anyway.
Afriocan agapanthus embryo callus after Afriocan agapanthus embryo callus, experimental group group 3 ultralow temperature after Afriocan agapanthus embryo callus, experimental group group 2 ultralow temperature after being preserved by left-to-right Afriocan agapanthus embryo callus, experimental group group 1 ultralow temperature being respectively after control group ultralow temperature is preserved in Fig. 1 are preserved are preserved.As shown in Figure 1, the larger expression fluorescence intensity of brightness is stronger, and cell viability is higher.
Embodiment
Below, by specific instantiation explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by the disclosed content of this specification.The present invention can also be implemented or be applied by other different embodiment, and the every details in this specification also can be based on different viewpoints and application, carries out various modifications or change not deviating under spirit of the present invention.
Afriocan agapanthus embryo callus used in the embodiment of the present invention is induced by bennet, concrete grammar is existing beautiful with reference to the research > > model of the blue Rapid Propagation of Lilium technology of < <, Shanghai Communications University's master thesis, 2009.
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, 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, the pH of described MS solid culture medium is 5.8.
3) pre-culture medium is the MS medium that contains 0.7mol/L sucrose.
4) loading liquid is: contain 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
5) vitrification solution is: contain 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 CNT.
6) cleaning solution is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
7) recovery media is the MS medium that contains 1.5mg/L picloram and 30g/L sucrose.
Embodiment
1) subculture is cultivated the Afriocan agapanthus embryo callus of 20 days on the MS solid culture medium that contains 0.5mol/L sucrose at 4 ℃ low temperature preculture 2 days;
2) going to soaking at room temperature in loading liquid processes 60 minutes;
3) proceed in vitrification solution under 0 ℃ of condition dehydration processing 40 minutes;
4) being finally placed in liquid nitrogen ultralow temperature preserves.
Step 3), after finishing, without removing vitrification solution, directly the Afriocan agapanthus embryo callus being soaked in vitrification solution is placed in to the preservation of liquid nitrogen ultralow temperature.
According to above-mentioned steps, Afriocan agapanthus embryo callus is divided into experimental group and control group.
Wherein, the graphene quantum dot that contains 0.1g/L, 0.3g/L, 0.5g/L in the vitrification solution of experimental group.
Particularly, the graphene quantum dot that contains 0.1g/L in the vitrification solution of experimental group group 1; The graphene quantum dot that contains 0.3g/L in the vitrification solution of experimental group group 2; The graphene quantum dot that contains 0.5g/L in the vitrification solution of experimental group group 3; In control group, difference is that vitrification solution does not contain graphene quantum dot, and other are identical with experimental group.
In liquid nitrogen, preserve after 1 hour and take out, put into fast 40 ℃ of water-baths, the 90s that thaws, and frequently shake gently; Vitrification solution is absorbed, added cleaning solution, room temperature treatment 30min, changes once washing liquid every 10min; Afriocan agapanthus embryo callus after washing moves on to recovery media and cultivates after 30 days, the relative survival rate of Afriocan agapanthus embryo callus in calculating comparative experiments group and control group.
The relative survival of the Afriocan agapanthus embryo callus of experimental group and control group is in Table 1.
Table 1
Experimental result
As shown in Table 1, the vitrification solution that the Afriocan agapanthus embryo callus ultralow temperature of the graphene quantum dot that employing contains 0.1g/L, 0.3g/L and 0.5g/L is preserved, makes Afriocan agapanthus embryo callus relative survival bring up to 68.34%, 72.34% and 60.87% by 53.42%.Wherein, best to contain 0.3g/L graphene quantum dot effect, concrete effect is shown in Fig. 1.
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 can, under spirit of the present invention and category, modify or change above-described embodiment.Therefore, such as in affiliated technical field, have and conventionally know that the knowledgeable, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
Claims (8)
1. a method of optimizing Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, is characterized in that, the method that adopts vitrification ultra-low temperature to preserve is preserved Afriocan agapanthus embryo callus, and concrete steps are as follows:
1) preculture: Afriocan agapanthus embryo callus is placed on pre-culture medium, and preculture is 1～4 day at 0～10 ℃;
2) loading liquid processes: under room temperature, Afriocan agapanthus embryo callus immersion treatment in loading liquid is removed to loading liquid after 40～60 minutes;
3) vitrification solution is processed: at 0～25 ℃, use vitrification solution to soak dehydration processing Afriocan agapanthus embryo callus 40～60 minutes;
4) liquid nitrogen is preserved: keep Afriocan agapanthus embryo callus to be immersed in the state of vitrification solution, and be placed in liquid nitrogen and preserve;
Described vitrification solution is the glass freezing protection liquid that contains 0.1～0.5g/L graphene quantum dot.
2. the method for optimizing as claimed in claim 1 Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, is characterized in that, described Afriocan agapanthus embryo callus pre-culture medium is the MS solid culture medium that contains 0.4～0.8mol/L sucrose.
3. the method for optimizing as claimed in claim 1 Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, is characterized in that, described loading liquid is for containing 1～2mol/L glycerine, 0.3～0.5mol/L sucrose and 5～10mmol/L KNO
3mS culture fluid.
4. optimize as claimed in claim 1 the method for Afriocan agapanthus embryo callus vitrification ultra-low temperature preservation effect, 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.
5. an Afriocan agapanthus embryo callus thaws and cultural method again, the Afriocan agapanthus embryo callus that described Afriocan agapanthus embryo callus is preserved for method as described in employing claim as arbitrary in claim 1～4, described Afriocan agapanthus embryo callus thaw and again cultural method for Afriocan agapanthus embryo callus is taken out from liquid nitrogen, first water-bath is thawed, then after removing vitrification solution, with cleaning solution, wash, finally proceed to renewal cultivation in recovery media.
6. thaw as claimed in claim 5 and cultural method again, it is characterized in that, the condition that water-bath is thawed for thawing 60～120s in the water-bath of 30～40 ℃.
7. thaw as claimed in claim 5 and cultural method again, it is characterized in that, described cleaning solution is for containing 1.0～1.5mol/L sucrose and 5～10mmol/L KNO
3mS culture fluid washing, washing process is: with cleaning solution, at room temperature Afriocan agapanthus embryo callus is soaked 10～30 minutes.
8. thaw as claimed in claim 5 and cultural method again, it is characterized in that, described recovery media is the MS medium that contains 1.0～3.0mg/L picloram and 30g/L sucrose.
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| CN108719278A (en) * | 2018-05-21 | 2018-11-02 | 上饶师范学院 | A method of improving early pears stem apex drop vitrification method cryopreservation effect |
| CN109479727A (en) * | 2019-01-22 | 2019-03-19 | 信阳农林学院 | A method of inducing cells,primordial using Afriocan agapanthus blade as explant |
| 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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| CN109479727A (en) * | 2019-01-22 | 2019-03-19 | 信阳农林学院 | A method of inducing cells,primordial using Afriocan agapanthus blade as explant |
| 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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