CN104170818B - A kind of method optimizing Agipanthus embryo callus vitrification ultra-low temperature preservation effect - Google Patents
A kind of method optimizing Agipanthus embryo callus vitrification ultra-low temperature preservation effect Download PDFInfo
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- CN104170818B CN104170818B CN201410468264.XA CN201410468264A CN104170818B CN 104170818 B CN104170818 B CN 104170818B CN 201410468264 A CN201410468264 A CN 201410468264A CN 104170818 B CN104170818 B CN 104170818B
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Abstract
The invention discloses a kind of method optimizing Agipanthus embryo callus vitrification ultra-low temperature preservation effect, for adopting the vitrification solution process hybrid cymbidium protocorms containing carbon nanomaterial to improve its preservation effect, specifically comprise: preculture, the process of loading liquid, vitrification solution process and Liquid nitrogen storage step, wherein said vitrification solution contains 0.1 ~ 0.5g/L graphene quantum dot.Method disclosed in the present invention is remarkable to the preservation effect optimization of Agipanthus embryo callus, plays promoter action by adding graphene quantum dot as allogenic material to the preservation of plant vitrification ultra-low temperature.
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 Agipanthus embryo callus vitrification ultra-low temperature preservation effect.
Background technology
Excised Embryos is the modern germ plasm resource in vitro conservation technology grown up the seventies in last century.Usually preserve in liquid nitrogen, be saved substance 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 perviousness and impermeability protective material; 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 vitreous 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.
Agipanthus calls blue lily, Afric lilium, is Agapanthaceae Agapanthus perennial plant, originates in south, Africa.Because its plant is tall and straight, leaf beautiful, leaf look dark green, flower amount is large, bright in luster and enjoy liking of people, has become common ornamental flower abroad, is used for garden cultivation and cut-flower production; In addition, Agipanthus also has that to fix the sand the Ecology Action and analgesia etc. of bank protection medicinal.The time that China introduces Agipanthus does not grow (2000), and it is low at the setting percentage in Shanghai, depend on external import seminal propagation, therefore, the demand of preservation to Agipanthus high quality seedling of the Vitro Quick Reproduction occurred by somatic embryo and Agipanthus germ plasm resource is most important more.Correlative study establishes Agipanthus embryo callus Excised Embryos system, and after preserving, the relative survival rate of cell reaches 56.94%, but still is not enough to the demand meeting 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 optimization Agipanthus embryo callus vitrification ultra-low temperature preservation effect newly, to overcome in prior art plant and to organize medium-term and long-term preservation to be difficult to realize, and the plant of Excised Embryos or the low shortcoming of its organized renewing growth rate.
To achieve these goals or other objects, the present invention is achieved by the following technical solutions.
Optimize a method for Agipanthus embryo callus vitrification ultra-low temperature preservation effect, the method adopting vitrification ultra-low temperature to preserve is preserved Agipanthus embryo callus, and concrete steps are as follows:
1) preculture: Agipanthus embryo callus is placed on pre-culture medium, preculture 1 ~ 4 day at 0 ~ 10 DEG C;
2) load liquid process: under room temperature, Agipanthus embryo callus immersion treatment in loading liquid was removed loading liquid after 40 ~ 60 minutes;
3) vitrification solution process: use vitrification solution to soak processed Agipanthus embryo callus 40 ~ 60 minutes at 0 ~ 25 DEG C;
4) Liquid nitrogen storage: keep the state that Agipanthus embryo callus is soaked by vitrification solution, and be placed in liquid nitrogen
Preserve;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot.
The nutrient solution 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 pyridoxine hydrochloride, 0.1mg/L vitamin, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/LZnSO
47H
2o, 0.25mg/L Na
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/LCoCl
26H
2o, surplus is water, and the pH of described MS nutrient solution is 5.8.
Preferably, described pre-culture medium is the MS solid medium containing 0.4 ~ 0.8mol/L sucrose.
Preferably, described pre-culture medium is the MS solid medium containing 0.5mol/L sucrose.
Preferably, described MS solid medium contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/LKH
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 pyridoxine hydrochloride, 0.1mg/L vitamin, 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 medium is 5.8.
Preferably, above-mentioned steps 1) described in Agipanthus embryo callus to be the method for cultivating on pre-culture medium be: Agipanthus embryo callus to be placed at 4 DEG C on pre-culture medium 1 ~ 4 day.
Preferably, above-mentioned steps 1) described in Agipanthus embryo callus to be the method for cultivating on pre-culture medium be: Agipanthus embryo callus is placed at 4 DEG C pre-culture medium upper 2 day.
Preferably, described loading liquid is for containing 1 ~ 2mol/L glycerol, 0.3 ~ 0.5mol/L sucrose and 5 ~ 10mmol/L KNO
3mS nutrient solution.
Preferably, described loading liquid is for containing 2mol/L glycerol, 0.4mol/L sucrose and 10mmol/L KNO
3mS nutrient solution.
Preferably, above-mentioned steps 2) in, under room temperature, Agipanthus embryo callus immersion treatment in loading liquid was removed loading liquid after 60 minutes;
Preferably, above-mentioned steps 3) in, at 0 DEG C, use vitrification solution to soak processed Agipanthus embryo callus 40 minutes.
Preferably, described vitrification solution is for containing 300g/L glycerol, 150g/L ethylene glycol, the MS nutrient solution 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 glycerol, 150g/L ethylene glycol, the MS nutrient solution 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 glycerol, 150g/L ethylene glycol, the MS nutrient solution 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 glycerol, 150g/L ethylene glycol, the MS nutrient solution 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 Agipanthus embryo callus, described Agipanthus embryo callus is the Agipanthus embryo callus adopting method as described above to preserve, described Agipanthus embryo callus thaw and again cultural method for Agipanthus embryo callus is taken out from liquid nitrogen, first water-bath is thawed, then wash with washings after removing vitrification solution, finally proceed to renewal cultivation in recovery media.
Preferably, the condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C.
More preferably, the condition that water-bath is thawed is the 90s that thaws in the water-bath of 40 DEG C.
More preferably, shake acceleration is adopted to thaw scheme when water-bath is thawed.
Preferably, described washings is for containing 1.0 ~ 1.5mol/L sucrose and 5 ~ 10mmol/L KNO
3mS nutrient solution.
Preferably, described washings is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS nutrient solution, washing process is: with washings at room temperature by Agipanthus embryo callus soak 10 ~ 30 minutes.
More preferably, described washing process is: at room temperature process 30 minutes with washings, changes once washing liquid every 10 minutes.
Preferably, described recovery media is the MS substratum containing 1.0 ~ 3.0mg/L picloram and 30g/L sucrose.
Preferably, described recovery media is the MS substratum containing 1.5mg/L picloram and 30g/L sucrose.
Method disclosed in the present invention is applicable to all Agipanthus embryo callus in prior art.Preferably, preparation method's reference of described Agipanthus 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 glycerol, 150g/L ethylene glycol, the MS nutrient solution of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.3g/L graphene quantum dot.
More preferably, relative survival is adopted to add up, particularly, utilize TTC (being abbreviated as TTC) legally constituted authority meter Agipanthus embryo callus relative survival after 30 days at renewal cultivation, and be aided with the observation of fluorescein diacetate (being abbreviated as FDA) staining.
According to nano science principle, in cryoprotectant, add viscosity and thermal conductivity that nano material effectively can improve cryoprotectant, change the formation situation of ice crystal, reduce injury to cell.The present invention preserves under vitrification ultra-low temperature condition Agipanthus embryo callus, first carry out preculture, then successively with loading liquid, vitrification solution process, wherein vitrification solution is again for the addition of the vitrification solution of graphene quantum dot, other techniques in the interpolation fitting method of this allogenic material, effectively can improve the preservation effect of Agipanthus embryo callus.According to store method disclosed by the invention, concentration is adopted to be that the graphene quantum dot of 0.1 ~ 0.5g/L is when using as allogenic material, recovery percentage after the preservation of Agipanthus embryo callus vitrification ultra-low temperature significantly improves, method disclosed in the present invention is remarkable to the preservation effect optimization of Agipanthus embryo callus, plays promoter action by adding graphene quantum dot as allogenic material to the preservation of plant vitrification ultra-low temperature.
Accompanying drawing explanation
Fig. 1 is the FDA stained photographs of Agipanthus embryo callus Excised Embryos restoration ecosystem in experimental group and control group.FDA can enter in viable cell protoplastis and produce fluorescence, can as judging cell mark anyway.
By the left-to-right Agipanthus embryo callus be respectively after the Agipanthus embryo callus after the Agipanthus embryo callus after control group Excised Embryos, experimental group group 1 Excised Embryos, the Agipanthus embryo callus after experimental group group 2 Excised Embryos, experimental group group 3 Excised Embryos in Fig. 1.As shown in Figure 1, brightness larger expression fluorescence intensity is stronger, and cell viability is higher.
Embodiment
Below by way of specific specific examples, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification sheets 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 sheets also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Agipanthus embryo callus used in the embodiment of the present invention is induced by bennet, concrete grammar reference " research of blue Rapid Propagation of Lilium technology " Fan Xianli, Shanghai Communications University's master thesis, 2009.
In the embodiment of the present invention, the formula of experiment reagent is as follows:
1) MS nutrient solution is: MS nutrient solution 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 pyridoxine hydrochloride, 0.1mg/L vitamin, 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/LNa
2moO
42H
2o, 0.025mg/L CuSO
45H
2o, 0.025mg/L CoCl
26H
2o, surplus is water, and the pH of described MS nutrient solution is 5.8.
2) MS solid medium is: MS solid medium contains 1900mg/L KNO
3, 1650mg/L NH
4nO
3, 170mg/LKH
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 pyridoxine hydrochloride, 0.1mg/L vitamin, 2mg/L glycine, 0.83mg/L KI, 6.2mg/L H
3bO
3, 22.3mg/L MnSO
44H
2o, 8.6mg/LZnSO
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 medium is 5.8.
3) pre-culture medium is the MS substratum containing 0.7mol/L sucrose.
4) loading liquid is: containing 2mol/L glycerol, 0.4mol/L sucrose and 10mmol/L KNO
3mS nutrient solution.
5) vitrification solution is: containing 300g/L glycerol, 150g/L ethylene glycol, the MS nutrient solution of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L carbon nanotube.
6) washings is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS nutrient solution.
7) recovery media is the MS substratum containing 1.5mg/L picloram and 30g/L sucrose.
Embodiment
1) by the succeeding transfer culture Agipanthus embryo callus of 20 days on the MS solid medium containing 0.5mol/L sucrose at 4 DEG C Low temperature preculture 2 days;
2) go to and to load in liquid soaking at room temperature process 60 minutes;
3) processed is proceeded in vitrification solution under 0 DEG C of condition 40 minutes;
4) liquid nitrogen Excised Embryos is finally placed in.
Step 3) terminate after, without the need to removing vitrification solution, directly the Agipanthus embryo callus be soaked in vitrification solution is placed in liquid nitrogen Excised Embryos.
According to above-mentioned steps, Agipanthus embryo callus is divided into experimental group and control group.
Wherein, the graphene quantum dot containing 0.1g/L, 0.3g/L, 0.5g/L in the vitrification solution of experimental group.
Particularly, the graphene quantum dot containing 0.1g/L in the vitrification solution of experimental group group 1; Graphene quantum dot containing 0.3g/L in the vitrification solution of experimental group group 2; Graphene quantum dot containing 0.5g/L in the vitrification solution of experimental group group 3; In control group, difference is vitrification solution not containing graphene quantum dot, and other are identical with experimental group.
Preserve in liquid nitrogen after 1 hour and take out, put into 40 DEG C of water-baths fast, thaw 90s, and frequently shake gently; Vitrification solution is absorbed, adds washings, room temperature treatment 30min, change once washing liquid every 10min; Agipanthus embryo callus after washing moves on to after recovery media cultivates 30 days, calculates and the relative survival rate of Agipanthus embryo callus in comparative experiments group and control group.
The relative survival of the Agipanthus embryo callus of experimental group and control group is in table 1.
Table 1
Experimental result
As shown in Table 1, adopt the vitrification solution of the Agipanthus embryo callus Excised Embryos of the graphene quantum dot containing 0.1g/L, 0.3g/L and 0.5g/L, make Agipanthus embryo callus relative survival bring up to 68.34%, 72.34% and 60.87% by 53.42%.Wherein, with best containing 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 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 (5)
1. optimize a method for Agipanthus embryo callus vitrification ultra-low temperature preservation effect, it is characterized in that, the method adopting vitrification ultra-low temperature to preserve is preserved Agipanthus embryo callus, and concrete steps are as follows:
1) preculture: Agipanthus embryo callus is placed on pre-culture medium, preculture 2 days at 4 DEG C;
2) load liquid process: under room temperature, Agipanthus embryo callus immersion treatment in loading liquid was removed loading liquid after 60 minutes;
3) vitrification solution process: use vitrification solution to soak processed Agipanthus embryo callus 40 minutes at 0 DEG C;
4) Liquid nitrogen storage: keep Agipanthus embryo callus to be immersed in the state of vitrification solution, and be placed in liquid nitrogen and preserve;
Described Agipanthus embryo callus is the succeeding transfer culture Agipanthus embryo callus of 20 days;
Described Agipanthus embryo callus pre-culture medium is the MS solid medium containing 0.7mol/L sucrose;
Described loading liquid is for containing 2mol/L glycerol, 0.4mol/L sucrose and 10mmol/L KNO
3mS nutrient solution;
Described vitrification solution is for containing 300g/L glycerol, 150g/L ethylene glycol, the MS nutrient solution of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
2. the thawing and cultural method again of an Agipanthus embryo callus, described Agipanthus embryo callus is the Agipanthus embryo callus adopting method as claimed in claim 1 to preserve, described Agipanthus embryo callus thaw and again cultural method for Agipanthus embryo callus is taken out from liquid nitrogen, first water-bath is thawed, then wash with washings after removing vitrification solution, finally proceed to renewal cultivation in recovery media.
3. thaw as claimed in claim 2 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.
4. thaw as claimed in claim 2 and cultural method again, it is characterized in that, described washings is for containing 1.0 ~ 1.5mol/L sucrose and 5 ~ 10mmol/L KNO
3mS nutrient solution washing, washing process is: with washings at room temperature by Agipanthus embryo callus soak 10 ~ 30 minutes.
5. thaw as claimed in claim 2 and cultural method again, it is characterized in that, described recovery media is the MS substratum containing 1.0 ~ 3.0mg/L picloram and 30g/L sucrose.
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| CN108719279B (en) * | 2018-05-21 | 2021-06-25 | 上饶师范学院 | Method for improving ultralow-temperature preservation effect of early pear stem tip embedding dehydration method |
| 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 |
| CN109479727B (en) * | 2019-01-22 | 2020-09-29 | 信阳农林学院 | Method for inducing embryonic cells by taking agapanthus praecox leaves as explants |
| CN110959330B (en) * | 2019-11-13 | 2021-07-13 | 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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