CN104255707A - Method for improving preservation effect of cymbidium type protocorm - Google Patents
Method for improving preservation effect of cymbidium type protocorm Download PDFInfo
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- CN104255707A CN104255707A CN201410467904.5A CN201410467904A CN104255707A CN 104255707 A CN104255707 A CN 104255707A CN 201410467904 A CN201410467904 A CN 201410467904A CN 104255707 A CN104255707 A CN 104255707A
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Abstract
The invention discloses a method for improving a preservation effect of cymbidium type protocorm. According to the invention, the cymbidium type protocorm is processed by adopting a vitrification solution which contains a carbon nanometer material so as to improve the preservation effect of the cymbidium type protocorm. The method specifically comprises the steps of pre-culture, contained liquid treatment, vitrification solution treatment and liquid nitrogen preservation, wherein the vitrification solution contains 0.1-0.5 g/L of carbon nano tubes. The method disclosed by the invention optimizes the preservation effect of the cymbidium type protocorm remarkably; as the carbon nano tubes 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 improving hybrid cymbidium protocorms 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.
The orchid family is the first section in angiosperm, as a kind of important ornamental plants resources, in the evolution of gardens and ornamental horticulture, occupies critical role.Hybrid cymbidium to being cultivated by artificial hybridization in Cymbidium, a general designation of the kind that beautiful in colour, flower is very large.At present, the orchid kind successfully realizing Excised Embryos comprises HERBA DENDROBII, dendrobium candidum, great Hua all ages orchid, the color stem of noble dendrobium, Platanthera, short all ages orchid etc.The relative survival of the hybrid cymbidium protocorms cryopreservation by vitrification system tentatively set up is only 10%, is also 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 raising hybrid cymbidium protocorms preservation effect newly, be difficult to realize to overcome the medium-term and long-term preservation of plant germplasm resource in prior art, 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.
Improve a method for hybrid cymbidium protocorms preservation effect, adopt the method for cryopreservation by vitrification to preserve hybrid cymbidium protocorms, concrete steps are as follows:
1) preculture: hybrid cymbidium protocorms is placed on pre-culture medium, preculture 1 ~ 5 day in illumination box;
2) load liquid process: under room temperature, hybrid cymbidium protocorms immersion treatment in loading liquid was removed loading liquid after 40 ~ 60 minutes;
3) vitrification solution process: use vitrification solution to soak dehydration processing hybrid cymbidium protocorms 40 ~ 60 minutes at 0 ~ 25 DEG C;
4) Liquid nitrogen storage: the state keeping hybrid cymbidium protocorms to soak in vitrification solution, is placed in liquid nitrogen and preserves;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L CNT.
Preferably, described 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.Preferably, the pH of described MS culture fluid is 5.8.
Preferably, above-mentioned steps 1) described in hybrid cymbidium protocorms be protocorms on the MS solid culture medium of the sorbierite containing 40 ~ 80g/L after illumination preculture.
Preferably, above-mentioned steps 1) described in hybrid cymbidium protocorms be protocorms on the MS solid culture medium of the sorbierite containing 60g/L after illumination preculture.
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/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 the method for cultivating on MS solid culture medium be: cultivate on the MS solid culture medium being placed in the sorbierite containing 60g/L under hybrid cymbidium protocorms room temperature 1 ~ 5 day.
Preferably, described loading liquid is for containing 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
More preferably, above-mentioned steps 2) in, under room temperature, hybrid cymbidium protocorms immersion treatment in loading liquid was removed loading liquid after 40 minutes;
More preferably, above-mentioned steps 3) in, at 0 DEG C, use vitrification solution to soak dehydration processing hybrid cymbidium protocorms 50 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 CNT.
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 CNT.
Thawing and cultural method again of a kind of hybrid cymbidium protocorms, described hybrid cymbidium protocorms is the hybrid cymbidium protocorms adopting method as described above to preserve, described hybrid cymbidium protocorms thaw and again cultural method for hybrid cymbidium protocorms 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 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 60s that thaws in the water-bath of 40 DEG C.
Preferably, described cleaning solution is for containing 1 ~ 1.5mol/L sucrose and 5 ~ 10mmol/L KNO
3mS culture fluid, washing process is: with cleaning solution at room temperature by hybrid cymbidium protocorms soak 10 ~ 30 minutes.
More preferably, described cleaning solution is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS culture fluid.Washing process is: at room temperature hybrid cymbidium protocorms is soaked 20 minutes with cleaning solution, changes cleaning solution every 10 minutes.
Preferably, described recovery media is the MS solid culture medium containing 1 ~ 2mg/L 6-benzyladenine, 0.1 ~ 0.5mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Preferably, described recovery media is the MS solid culture medium containing 2mg/L 6-benzyladenine, 0.1mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Preferably, preparation method's bibliography of hybrid cymbidium protocorms: hybrid cymbidium ' phantom ' tissue culture regeneration Establishing and Vitro Preservation (Liu Peipei, Beijing Forestry University's master thesis, 2008).
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 CNT.
More preferably, described washing process is: at room temperature soak 20 minutes with cleaning solution, within every 10 minutes, changes once washing liquid.
More preferably, in order to prove the preservation effect of method in the present invention, relative survival is adopted to add up, particularly, utilize TTC (being abbreviated as TTC) legally constituted authority meter hybrid cymbidium protocorms relative survival after 30 days at renewal cultivation, 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 thermal conductivity that nano material effectively can improve cryoprotector, change the formation situation of ice crystal, reduce injury to cell.The present invention preserves under vitrification ultra-low temperature condition hybrid cymbidium protocorms, first carry out preculture, then successively with loading liquid, vitrification solution process, finally Excised Embryos in liquid nitrogen, wherein vitrification solution is again for the addition of the vitrification solution of CNT, other techniques in the interpolation fitting method of this allogenic material, effectively can improve the preservation effect of hybrid cymbidium protocorms.According to store method disclosed by the invention, concentration is adopted to be that the CNT of 0.1 ~ 0.5g/L is when using as allogenic material, relative survival after the preservation of hybrid cymbidium protocorms vitrification ultra-low temperature significantly improves, method disclosed in the present invention is remarkable to the preservation effect optimization of hybrid cymbidium protocorms, plays facilitation by adding CNT as allogenic material to the preservation of plant vitrification ultra-low temperature.
Accompanying drawing explanation
Fig. 1 is the FDA stained photographs of hybrid cymbidium protocorms Excised Embryos restoration ecosystem in experimental group and control group in embodiment 1.FDA can enter in living cells protoplast and produce fluorescence, can as judging cell mark anyway.
By the left-to-right hybrid cymbidium protocorms be respectively after the hybrid cymbidium protocorms after the hybrid cymbidium protocorms after control group Excised Embryos, experimental group group 1 Excised Embryos, the hybrid cymbidium protocorms 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 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.
Hybrid cymbidium protocorms used in the embodiment of the present invention by Stem tip induction, concrete grammar with reference to " hybrid cymbidium ' phantom ' tissue culture regeneration Establishing and Vitro Preservation ", Liu Peipei, Beijing Forestry University's master thesis, 2008.
In the embodiment of the present invention, the formula of experiment reagent used 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) pre-culture medium is the MS medium containing 60g/L sorbierite.
4) loading liquid is: containing 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
5) 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 CNT.
6) cleaning solution is for containing 1.2mol/L sucrose and 10mmol/L KNO
3mS culture fluid.
7) recovery media is the MS solid culture medium containing 2mg/L 6-benzyladenine, 0.1mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Embodiment 1
1) by hybrid cymbidium protocorms illumination preculture 5 days on the MS solid culture medium containing 60g/L sorbierite;
2) go to and to load in liquid soaking at room temperature process 40 minutes;
3) dehydration processing is proceeded in vitrification solution under 0 DEG C of condition 50 minutes;
4) liquid nitrogen Excised Embryos is finally placed in.
Step 3) terminate after, without the need to removing vitrification solution, directly the hybrid cymbidium protocorms be soaked in vitrification solution is placed in liquid nitrogen Excised Embryos.
According to above-mentioned steps, hybrid cymbidium protocorms is divided into experimental group and control group.
CNT respectively containing 0.1g/L, 0.3g/L, 0.5g/L in the vitrification solution of experimental group.
CNT containing 0.1g/L in the vitrification solution of experimental group group 1; CNT containing 0.3g/L in the vitrification solution of experimental group group 2; CNT containing 0.5g/L in the vitrification solution of experimental group group 3; In control group, difference is that vitrification solution is not added with CNT, 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 60s, and frequently shake gently; Vitrification solution is absorbed, adds cleaning solution, room temperature washing 20min, change once washing liquid every 10min; Hybrid cymbidium protocorms after washing moves on to after recovery media cultivates 30 days, calculates and the relative survival rate of hybrid cymbidium protocorms in comparative experiments group and control group.
The relative survival rate of the hybrid cymbidium protocorms of experimental group and control group is in table 1.
Table 1
Experimental result
As shown in Table 1, be that the CNT of 0.1g/L, 0.3g/L and 0.5g/L is present in the vitrification solution of hybrid cymbidium protocorms Excised Embryos by concentration, make hybrid cymbidium protocorms recovery percentage bring up to 20.89%, 16.75% and 10.78% by 8.85%.Wherein, the most remarkable with the raising effect of adding 0.1g/L CNT, 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 (8)
1. improve a method for hybrid cymbidium protocorms preservation effect, it is characterized in that, adopt the method for cryopreservation by vitrification to preserve hybrid cymbidium protocorms, concrete steps are as follows:
1) preculture: hybrid cymbidium protocorms is placed on pre-culture medium, preculture 1 ~ 5 day in illumination box;
2) load liquid process: under room temperature, hybrid cymbidium protocorms immersion treatment in loading liquid was removed loading liquid after 40 ~ 60 minutes;
3) vitrification solution process: use vitrification solution to soak dehydration processing hybrid cymbidium protocorms 40 ~ 60 minutes at 0 ~ 25 DEG C;
4) Liquid nitrogen storage: the state keeping hybrid cymbidium protocorms to soak in vitrification solution, is placed in liquid nitrogen and preserves;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L CNT.
2. improve the method for hybrid cymbidium protocorms preservation effect as claimed in claim 1, it is characterized in that, described hybrid cymbidium protocorms pre-culture medium is the MS solid culture medium containing 40 ~ 80g/L sorbierite.
3. improve the method for hybrid cymbidium protocorms preservation effect as claimed in claim 1, it 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. improve the method for hybrid cymbidium protocorms 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 CNT.
5. the thawing and cultural method again of a hybrid cymbidium protocorms, described hybrid cymbidium protocorms is for adopting the hybrid cymbidium protocorms that as described in claim as arbitrary in Claims 1 to 4, method is preserved, described hybrid cymbidium protocorms thaw and again cultural method for hybrid cymbidium protocorms 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 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 is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C.
7. thaw as claimed in claim 5 and cultural method again, it is characterized in that, described cleaning solution is for containing 1 ~ 1.5mol/L sucrose and 5 ~ 10mmol/L KNO
3mS culture fluid washing, washing process is: with cleaning solution at room temperature by hybrid cymbidium protocorms soak 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 solid culture medium containing 1 ~ 2mg/L 6-benzyladenine, 0.1 ~ 0.5mg/L methyl α-naphthyl acetate and 30g/L sucrose.
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Cited By (6)
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| CN106718876A (en) * | 2016-11-23 | 2017-05-31 | 中国科学院华南植物园 | The Cord blood culture medium and Cord blood propagation method of a kind of Helen's pocket orchid protocorm |
| CN106937592A (en) * | 2016-10-27 | 2017-07-11 | 中国科学院昆明植物研究所 | A kind of Tibet Cymbidium hookerianum stem apex droplet cryopreservation by vitrification method |
| CN108719279A (en) * | 2018-05-21 | 2018-11-02 | 上饶师范学院 | A method of improving early pears stem apex Encapsulation dehydration cryopreservation effect |
| 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 |
| CN108782238A (en) * | 2018-05-03 | 2018-11-13 | 上饶师范学院 | A kind of method of morning pears stem with bud Plantlet in vitro |
| CN108812308A (en) * | 2018-05-21 | 2018-11-16 | 上饶师范学院 | A method of improving early pears stem apex encapsulation- vitrification method cryopreservation effect |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106937592A (en) * | 2016-10-27 | 2017-07-11 | 中国科学院昆明植物研究所 | A kind of Tibet Cymbidium hookerianum stem apex droplet cryopreservation by vitrification method |
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| CN108719279A (en) * | 2018-05-21 | 2018-11-02 | 上饶师范学院 | A method of improving early pears stem apex Encapsulation dehydration cryopreservation effect |
| 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 |
| CN108812308A (en) * | 2018-05-21 | 2018-11-16 | 上饶师范学院 | A method of improving early pears stem apex encapsulation- vitrification method cryopreservation effect |
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