CN111387176A - Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus - Google Patents
Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus Download PDFInfo
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- CN111387176A CN111387176A CN202010333403.3A CN202010333403A CN111387176A CN 111387176 A CN111387176 A CN 111387176A CN 202010333403 A CN202010333403 A CN 202010333403A CN 111387176 A CN111387176 A CN 111387176A
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- 238000004017 vitrification Methods 0.000 title claims abstract description 36
- 238000004321 preservation Methods 0.000 title claims abstract description 30
- 241000196324 Embryophyta Species 0.000 claims abstract description 14
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- 230000000408 embryogenic effect Effects 0.000 claims description 26
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- 230000002401 inhibitory effect Effects 0.000 description 1
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- 229960000367 inositol Drugs 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a vitrification ultralow temperature preservation method for magnolia officinalis embryonic callus, and relates to the technical field of magnolia officinalis germplasm preservation. The method is based on the characteristics that the proliferation rate of the regenerated cortex magnoliae officinalis embryonic callus is high, the proliferation and differentiation can be carried out for a long time, the efficiency of differentiation into somatic embryos is high, and the efficiency of transformation into plants is high, and the loading treatment and plant vitrification treatment are carried out on the embryonic callus so as to finish the ultralow-temperature preservation. The method has the advantages of permanent storage, no influence of natural environment change, high storage efficiency and convenient management; and after regeneration, proliferation and differentiation can be rapidly carried out, the differentiated somatic embryos directly develop into plants, the conversion rate is high, and rooting culture is not needed. The regeneration rate of the ultralow temperature preservation is 100%, and the ultralow temperature preservation method can be used for carrying out the ultralow temperature preservation on the magnolia officinalis germplasm resources for a long time.
Description
Technical Field
The invention belongs to the technical field of preservation of magnolia germplasm, and particularly relates to a vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus.
Background
Cortex Magnolia officinalis (Magnolia officinalis of Magnolia genus of Magnoliaceae family) bark, root bark, branch bark and flower and fruit can be used as raw materials, the bark is a famous Chinese medicine, and has a medicinal history of more than two thousand years in China, the main components of cortex Magnolia officinalis include magnolol, honokiol, magnoline, volatile oil, β -eudesmol, etc., wherein the main component is magnolol (C)18H18O2) With honokiol (C)18H18O2) Has antitumor, antibacterial, antiulcer, antidepressant, and anticarious effects. Magnolol has inhibitory effect on various cancer cells, and can be used for treating depression.
The magnolia officinalis has not only important medicinal value but also higher ornamental value. Because the bark can be used with drugs, the over-cutting is caused, the resources and the distribution are sharply reduced, and the bark is listed as a national secondary protection wild plant. Therefore, the preservation of the magnolia bark germplasm resources becomes the key for the sustainable utilization of the magnolia bark. At present, the germplasm resources of mangnolia officinalis are mainly preserved by establishing a protective area and artificially planting.
Disclosure of Invention
In view of the above, the invention aims to provide a vitrification cryopreservation method for magnolia bark embryonic callus, which is simple and easy to implement, high in stability, high in efficiency and reliability, and can be used for rapidly differentiating a large number of magnolia bark somatic embryos after preservation by utilizing the differentiation capability of the embryonic callus, and further developing into complete plants with good growth state after transplantation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a vitrification ultra-low temperature preservation method of magnolia officinalis embryonic callus, which comprises the following steps of (1) mixing the magnolia officinalis embryonic callus with a loading solution for loading treatment to obtain the loading callus, wherein the loading solution consists of WPM basal medium, 184 g/L glycerol and 136.8 g/L sucrose;
(2) mixing the loaded callus with a vitrifying solution 2solution of ice for vitrifying to obtain a vitrified callus, wherein the vitrifying solution 2solution consists of a WPM basal culture medium, 300 g/L glycerol, 150 g/L ethylene glycol, 150 g/L dimethyl sulfoxide and 0.4 mol/L sucrose;
(3) and (3) preserving the vitrified callus in liquid nitrogen.
Preferably, the method for preparing the magnolia officinalis embryonic callus in the step (1) comprises the steps of carrying out induction culture on magnolia officinalis seeds in an induction culture medium for 2 weeks, wherein the induction culture medium consists of a WPM basic culture medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1 g/L polyvinylpyrrolidone, 1 g/L hydrolyzed casein, 40 g/L sucrose and 3 g/L plant gel, and the pH value is 5.8;
preferably, the induction culture is a dark culture, and the temperature of the induction culture is 25 ℃.
Preferably, the volume ratio of the magnolia embryogenic callus to the loading solution during the mixing in the step (1) is 1: 8.
Preferably, before the mixing in the step (2), the method further comprises the step of sucking and discarding the loading solution; and (3) during the mixing in the step (2), the volume ratio of the loading callus to the vitrification solution 2 is 1: 8.
Preferably, the vitrification treatment in the step (2) is performed on ice, and the vitrification treatment time is 30 min.
Preferably, after the preservation in step (3), restoring the preserved callus is further included.
Preferably, the storage time is at least 24 h.
Preferably, the recovery method comprises the following steps: (a) unfreezing the preserved callus in a water bath at 40 ℃;
(b) sucking and discarding the vitrification solution 2solution in the thawed callus, mixing the thawed callus with unloading solution, and unloading to obtain unloaded callus, wherein the unloading solution consists of WPM basal medium and 410.4 g/L sucrose, and has pH of 5.8;
(c) after the unloading solution was aspirated off, the unloaded calli were transferred to the induction medium for recovery culture.
Preferably, before the transferring in the step (c), the method further comprises washing the unloaded callus with a WPM liquid medium, mixing the washed unloaded callus with the WPM liquid medium, and blotting the WPM liquid medium with filter paper.
Compared with the prior art, the invention has the following beneficial effects: the vitrified ultralow temperature preservation method for the embryonic callus of the magnolia officinalis is based on the characteristics that the proliferation rate of the regenerated embryonic callus of the magnolia officinalis is high, the proliferation and differentiation can be carried out for a long time, the efficiency of differentiation into somatic embryos is higher, and the efficiency of transformation into plants is high, the embryonic callus is used for loading treatment, and the plant vitrification treatment is carried out, so that the ultralow temperature preservation is completed; the loading treatment and the vitrification treatment aim to reduce the water content in the cells and increase the penetration type in the cells, so that the icing in the cells can be reduced when the cells are put into liquid nitrogen, and the integrity of cell membranes can be further protected.
Compared with the existing in-situ established protection area storage and ex-situ storage, the method has the advantages of permanent storage, no influence of natural environment change, high storage efficiency and convenience in management. Compared with the existing tissue culture and preservation, the method has the advantages of high genetic stability, high preservation efficiency, rapidness, convenience, rapidness, proliferation and differentiation after regeneration, direct development of differentiated somatic embryos into plants, high conversion rate and no need of rooting culture. The regeneration rate of the ultralow temperature preservation is 100%, and the ultralow temperature preservation method can be used for carrying out the ultralow temperature preservation on the magnolia officinalis germplasm resources for a long time.
Drawings
FIG. 1 shows the survival rate of embryogenic callus of Magnolia officinalis after cryopreservation;
FIG. 2 is a recovery process of ultralow temperature preservation of callus of Magnolia officinalis embryo;
FIG. 3 shows the differentiation of the regenerated embryogenic callus into seedlings.
Detailed Description
The invention provides a vitrification ultra-low temperature preservation method of magnolia officinalis embryonic callus, which comprises the following steps of (1) mixing the magnolia officinalis embryonic callus with a loading solution for loading treatment to obtain the loading callus, wherein the loading solution consists of WPM basal medium, 184 g/L glycerol and 136.8 g/L sucrose;
(2) mixing the loaded callus with a vitrifying solution 2solution of ice for vitrifying to obtain a vitrified callus, wherein the vitrifying solution 2solution consists of a WPM basal culture medium, 300 g/L glycerol, 150 g/L ethylene glycol, 150 g/L dimethyl sulfoxide and 0.4 mol/L sucrose;
(3) and (3) preserving the vitrified callus in liquid nitrogen.
In the method, magnolia officinalis embryogenic callus is mixed with loading solution (loading solution) for loading treatment to obtain loaded callus, wherein the L loading solution consists of WPM basal medium, 184 g/L glycerol and 136.8 g/L sucrose, the preparation method of the magnolia officinalis embryogenic callus preferably comprises the following steps of carrying out induction culture on magnolia officinalis seeds in an induction culture medium for 2 weeks, wherein the induction culture medium consists of WPM basal medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1 g/L polyvinylpyrrolidone, 1 g/L hydrolyzed casein, 40 g/L sucrose and 3 g/L plant gel, the pH is 5.8, the source of the magnolia officinalis seeds is not particularly limited, the callus induction is preferably carried out by taking mature seeds as materials, the induction culture is preferably carried out in a dark, the temperature of the dark culture is preferably 25 ℃, the preparation method of the induction culture medium is preferably carried out by the method, the preparation method is not particularly limited, the composition of the culture medium is adjusted after the WPM is adjusted, and the composition is 1.8:
TABLE 1WPM basal Medium composition
Serial number | Compound (I) | Concentration mg/ |
1 | NH4NO3 | 400 |
2 | CaCl2·2H2O | 96 |
3 | Ca(NO3)2·4H2O | 556 |
4 | MgSO4·7H2O | 370 |
5 | K2SO4 | 990 |
6 | KH2PO4 | 170 |
7 | H3BO3 | 602 |
8 | CuSO4·5H2O | 0.25 |
9 | MnSO4·H2O | 22.3 |
10 | Na2Mo4·2H2O | 0.25 |
11 | ZnSO4·7H2O | 8.6 |
12 | Na2EDTA | 37.3 |
13 | FeSO4·7H2O | 27.8 |
14 | Myo- |
100 |
When the method is used for mixing, the method preferably further comprises the steps of measuring a certain amount of the embryogenic callus, taking 0.25m L as an example, preferably adding 1m L WPM liquid culture medium into a sterilized 1.5m L centrifuge tube, and measuring the Magnolia cortex embryogenic callus with the volume of 0.25m L.
The volume ratio of the magnolia officinalis embryogenic callus to the loading solution is preferably 1:8 during the mixing, the loading treatment is carried out after the mixing, the loading treatment temperature is preferably 25 ℃, the loading treatment time is preferably 20min, the preparation method of the L loading solution is not particularly limited, the components are preferably mixed, the pH value is adjusted to 5.8, and the preparation method further comprises the steps of carrying out high-pressure damp-heat sterilization at 121 ℃ for 15min and standing to normal temperature.
After obtaining the loaded callus, the loaded callus is mixed with an ice vitrification solution 2solution (PVS2solution) to be vitrified, so as to obtain vitrified callus, wherein the vitrification solution 2solution (PVS2solution) consists of a WPM basic medium, 300 g/L glycerol, 150 g/L ethylene glycol, 150 g/L dimethyl sulfoxide and 0.4 mol/L sucrose, before the mixing, the method preferably further comprises sucking and discarding the loading solution (loading solution), when the mixing is carried out, the volume ratio of the loaded callus to the PVS2solution is preferably 1:8, the vitrification treatment is carried out on ice, the vitrification treatment is preferably 30min, a preparation method for the PVS2solution is not particularly limited, after the components are mixed, the pH value is adjusted to 5.8, the temperature is 121 ℃, the high-pressure moist heat sterilization is carried out, then the vitrification solution is kept to room temperature, and the vitrification solution is preferably carried out after the PVS 678678, and when the vitrification solution is pre-frozen, the vitrification solution is preferably carried out, and the vitrification solution is carried out on the PVS 678678.
After the vitrified callus is obtained, the vitrified callus is placed in liquid nitrogen for preservation. In the preservation of the invention, preferably, the mixed solution of the vitrified callus and the PVS2solution is placed in a freezing tube, fixed on a bracket of the freezing tube and placed in a liquid nitrogen tank for preservation.
After the preservation, the invention preferably also comprises restoring the preserved callus; the storage time is preferably at least 24 h.
The recovery method of the present invention preferably comprises the following steps: (a) unfreezing the preserved callus in a water bath at 40 ℃;
(b) absorbing PVS2solution in the thawed callus, mixing the thawed callus with Unloading solution (Unloading solution) to obtain unloaded callus, wherein the Unloading solution (Unloading solution) consists of WPM basal medium and 410.4 g/L of cane sugar, and has the pH value of 5.8;
(c) after the Unloading solution (Unloading solution) was aspirated, the unloaded calli were transferred to the induction medium for recovery culture.
In the invention, when the water bath in step (a) is carried out, the cryopreservation bracket attached with the cryopreservation tube is preferably rapidly inserted into a constant-temperature water bath at 40 ℃, and the thawing time of the water bath is preferably 2 min.
Before the unloading treatment in the step (b), the method preferably further comprises sterilizing the surface of the cryopreserved pipe after the water bath, preferably wiping the surface with 75% alcohol by volume fraction, and then placing the surface in an ultra-clean bench for the unloading treatment. The addition amount of the Unloading solution is preferably the same as that of the loading solution. The unloading treatment according to the invention is preferably carried out at 25 ℃ for 20 min. The preparation method of the Unloading solution is not particularly limited in the invention, and the Unloading solution is preferably prepared by mixing the components, adjusting the pH to 5.8, and then carrying out high-pressure moist heat sterilization at 121 ℃ for 15min and standing to normal temperature.
After the unloading treatment result and before the transfer, the invention preferably also comprises the steps of washing the unloaded callus by using a WPM liquid culture medium, mixing the washed unloaded callus with the WPM liquid culture medium, and sucking the WPM liquid culture medium by using filter paper. The filter paper of the present invention is preferably 3 pieces of filter paper stacked and placed in a 9cm glass petri dish. The recovery culture is preferably carried out at 25 ℃ under the dark condition to obtain the regenerative embryogenic callus.
After the regenerative embryogenic callus is obtained, the method preferably further comprises the steps of subculture, differentiation and seedling formation of the regenerative embryogenic callus, and more preferably, after the 1-month recovery culture, the embryogenic callus regenerated on the filter paper is transferred to the induction medium without the filter paper, and is subjected to subculture under the dark condition at 25 ℃. In the subculture process, the culture medium is replaced every two weeks. The invention preferably selects embryogenic callus of two weeks after two subcultures, transfers the embryogenic callus to WPM culture medium to be differentiated, after 1 month, there is somatic embryo generated without callus, picks out the somatic embryo, places the somatic embryo on WPM culture medium, transfers the somatic embryo to tissue culture bottle for continuous culture after root and cotyledon begin to grow out, transfers the root system to seedling culture hole after the root system is sent out, finally makes bag seedling. The WPM culture medium is preferably prepared by adding 1% (mass ratio) of agar to the WPM basic culture medium, dissolving, and sterilizing.
The vitrification cryopreservation method of magnolia embryogenic callus provided by the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. The material is selected from Magnolia officinalis embryonic callus induced by mature seed zygote embryo of Magnolia officinalis, and Magnolia officinalis embryonic callus cultured in WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1 g/L PVP (polyvinylpyrrolidone) +1 g/L hydrolyzed Casein (CH) +40 g/L sucrose +3 g/L plant gel under the conditions of pH5.8 and 25 deg.C in dark for 2 weeks.
2. Measuring 0.25m L cortex Magnolia officinalis embryogenic callus by adding 1m L WPM culture medium into sterilized 1.5m L centrifuge tube in an ultra-clean bench, wherein the scale is equal to the lowest part of 1m L concave liquid level, picking cortex Magnolia officinalis embryogenic callus with good growth state with forceps, transferring into 1.5m L centrifuge tube until the lowest part of concave liquid level is equal to the scale of 1.25m L, and measuring cortex Magnolia officinalis embryogenic callus with volume of 0.25m L.
3. And (3) loading treatment, namely, installing a 1m L-cut blue gun head on a pipette gun, transferring the weighed 0.25m L embryonic callus into a 2m L freezing storage tube, completely sucking up WPM liquid, and adding 2m L loading solution for loading treatment for 20 minutes.
4. Vitrification treatment, namely completely absorbing the loading liquid by using a liquid transfer gun after the loading treatment is finished, adding 2m L of pre-cooled PVS2solution on ice, and treating for 30 minutes on the ice.
5. And (4) ultra-low temperature preservation: and (5) after the vitrification treatment is finished, freezing and fixing the freezing and storing pipe on the freezing and storing pipe bracket. And (5) rapidly placing the freezing tube bracket into a liquid nitrogen tank for storage for more than 24 hours.
6. Thawing in water bath at 40 ℃: and (3) taking the cryopreservation bracket attached to the cryopreservation tube after being preserved in liquid nitrogen for more than 24 hours out of the liquid nitrogen tank, quickly inserting the bracket into a constant-temperature water bath at 40 ℃, and unfreezing the bracket in the water bath for 2 minutes.
7. Unloading, namely freezing and storing the tube after water bath, wiping the outer surface of the tube with 75% alcohol, completely sucking PVS2solution in an ultra-clean workbench by using a liquid transfer gun, adding 2m L Unloading solution, and treating for 20 minutes at 25 ℃;
8. cleaning and recovery culturing, namely after Unloading treatment, completely sucking the Unloading solution by using a pipette gun, adding 2m L WPM liquid culture medium for cleaning, completely sucking the WPM liquid by using the pipette gun, adding 1m L WPM liquid into a 2m L freezing storage tube after cleaning, installing a cutting gun head by using the pipette gun, transferring the embryonic callus into a 9cm glass culture dish containing 3 pieces of filter paper, completely permeating the filtrate, transferring to WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1 g/L PVP +1 g/L CH +30 g/L sucrose +3 g/L Phytagel (plant gel), and performing recovery culturing under the dark condition of pH5.8 and 25 ℃.
The 190-tube embryogenic callus after two weeks of freezing was subjected to recovery culture 5 times, and the results are shown in FIG. 1, wherein 40 tubes were recovered at 1 st, 2 nd, 3 nd and 4 th times, and 30 tubes were recovered at 5 th time, and the survival rate after recovery was 100%.
9. Identification of survival rate and recovery ability and selection of optimal vitrification solution treatment time
The fluorescent change of the magnolia officinalis embryonic callus at the recovery time of 0 hour, 1 day, 2 days, 3 days, 4 days, 5 days and 6 days is observed by a laser confocal microscope after the fluorescent dye of the Fluorescein Diacetate (FDA) live cells and the fluorescent dye of the Propidium Iodide (PI) dead cells are used for dyeing, and the result shows that the viability value is firstly reduced in the recovery culture process, the cell viability value at 48 hours is the lowest, and then the cell viability value is increased at 7 days, which is close to the control. Thereby obtaining the most accurate time point for measuring the activity rate of the embryogenic callus of the magnolia officinalis within 48 hours.
And then the PVS2 vitrification solution is treated for 0 minute, 5 minutes, 10 minutes, 30 minutes, 50 minutes, 70 minutes and 90 minutes under the time gradient, the ultra-low temperature storage is carried out, the fluorescence and the fluorescence intensity after 48 hours of culture are recovered, and the result is recovered for 48 hours, and the PVS2 vitrification solution is treated for 30 minutes to obtain the highest fluorescence intensity, namely the highest activity. The observation of the ultra-low temperature embryogenic callus at two weeks (as shown in FIG. 2, wherein a is the raw material for ultra-low temperature preservation for two weeks, b is the callus of Magnolia officinalis embryo which is thawed and then restored for 48 hours, c is the callus of Magnolia officinalis embryo which is thawed and then restored for 2 weeks, and d is the callus of Magnolia officinalis embryo which is ultra-low temperature regeneration for two weeks) is combined to obtain 30 minutes which is the most vitrification time of the callus of Magnolia officinalis embryo.
10. Subculture, differentiation and seedling formation of regenerated embryonic callus
When the regeneration culture is carried out for 1 month, transferring the embryogenic callus regenerated on the filter paper to a tissue culture medium which is not added with the filter paper, wherein the tissue culture medium comprises WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1 g/L PVP +1 g/L CH +30 g/L sucrose +3 g/L Phytagel (plant gel), the temperature is 5.8, and the temperature is 25 ℃ under dark condition, carrying out subculture once every two weeks, selecting embryogenic tissue subculturing two weeks, transferring to a WPM medium to differentiate, generating somatic embryos after 1 month without callus, picking out the somatic embryos, placing the somatic embryos on the WPM medium, continuing to culture after roots and cotyledons begin to grow, transferring to a bottle to grow into a seedling raising hole, finally making into a bag seedling, and recovering the seedling process is shown in figure 3, wherein a is the embryogenic callus regenerated by freezing, b is the embryogenic callus regenerated tissue culture medium, the tissue culture medium is the callus tissue culture medium, the tissue culture medium is transferred to a seedling raising hole, and the survival rate is 100 cm.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A vitrification cryopreservation method for callus of magnolia officinalis embryo is characterized in that the vitrification cryopreservation method comprises the following steps of (1) mixing the callus of magnolia officinalis embryo with loading solution to obtain the loading callus, wherein the loading solution consists of WPM basal medium, 184 g/L glycerol and 136.8 g/L sucrose;
(2) mixing the loaded callus with a vitrifying solution 2solution of ice for vitrifying to obtain a vitrified callus, wherein the vitrifying solution 2solution consists of a WPM basal culture medium, 300 g/L glycerol, 150 g/L ethylene glycol, 150 g/L dimethyl sulfoxide and 0.4 mol/L sucrose;
(3) and (3) preserving the vitrified callus in liquid nitrogen.
2. The method of claim 1, wherein the callus derived from magnolia officinalis embryos obtained in step (1) is obtained by inducing magnolia officinalis seeds in an induction medium consisting of WPM basal medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1 g/L polyvinylpyrrolidone, 1 g/L hydrolysed casein, 40 g/L sucrose and 3 g/L plant gel, and pH5.8, for 2 weeks.
3. The method of claim 2, wherein the induction culture is a dark culture and the temperature of the induction culture is 25 ℃.
4. The method of claim 1, wherein the mixing of step (1) is performed in a 1:8 ratio of the embryogenic callus of Magnolia officinalis to the loading solution.
5. The method of claim 1, wherein prior to said mixing in step (2), further comprising the steps of discarding the loading solution; and (3) during the mixing in the step (2), the volume ratio of the loading callus to the vitrification solution 2 is 1: 8.
6. The method according to claim 1, wherein the vitrification treatment of the step (2) is performed on ice, and the vitrification treatment time is 30 min.
7. The method of any one of claims 1 to 6, further comprising recovering the callus after the preservation in step (3).
8. The method of claim 7, wherein the holding time is at least 24 hours.
9. The method of claim 7, wherein the recovering method comprises the steps of: (a) unfreezing the preserved callus in a water bath at 40 ℃;
(b) sucking and discarding the vitrification solution 2solution in the thawed callus, mixing the thawed callus with unloading solution, and unloading to obtain unloaded callus, wherein the unloading solution consists of WPM basal medium and 410.4 g/L sucrose, and has pH of 5.8;
(c) after the unloading solution was aspirated off, the unloaded calli were transferred to the induction medium for recovery culture.
10. The method of claim 9, further comprising washing said unloaded callus with WPM broth, mixing said washed unloaded callus with WPM broth, and blotting said WPM broth with filter paper prior to said transferring in step (c).
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