CN111387176B

CN111387176B - Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus

Info

Publication number: CN111387176B
Application number: CN202010333403.3A
Authority: CN
Inventors: 林亮; 贾艳霞; 马俊超; 郁步竹; 徐倩; 高彦粉; 李唯奇
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2021-07-30
Anticipated expiration: 2040-04-24
Also published as: CN111387176A

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

Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus

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

Magnolia officinalis (Magnolia officinalis of rhesus rehd. etwils) belongs to the Magnolia (Magnolia) plant of the magnoliaceae family (Magnolia acejuss). Cortex Magnoliae officinalis, root bark, branch bark, flower and fruit can be used as the medicine, and bark is a famous traditional Chinese medicine and has been used for more than two thousand years in China. The main components of cortex Magnolia officinalis include magnolol, honokiol, magnoline, volatile oil, and beta-eudesmol, 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 ultralow temperature preservation method for magnolia officinalis embryonic callus, which comprises the following steps: (1) mixing the cortex magnoliae officinalis embryonic callus with a loading solution for loading treatment to obtain loaded callus; the loading solution consists of WPM basal medium, 184g/L of glycerol and 136.8g/L of sucrose;

(2) mixing the loaded callus with a vitrification solution 2 of ice for vitrification treatment to obtain vitrified callus; the vitrification solution 2 solution consists of a WPM basic culture medium, 300g/L of glycerol, 150g/L of ethylene glycol, 150g/L of dimethyl sulfoxide and 0.4mol/L of cane sugar;

(3) and (3) preserving the vitrified callus in liquid nitrogen.

Preferably, the method for preparing magnolia bark embryonic callus in the step (1) comprises the steps of inducing and culturing magnolia bark seeds in an induction culture medium for 2 weeks; the induction culture medium consists of a WPM basic culture medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1g/L polyvinylpyrrolidone, 1g/L casein hydrolysate, 40g/L sucrose and 3g/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 2 in the thawed callus, mixing the thawed callus with an unloading solution, and then carrying out unloading treatment to obtain unloaded callus; the unloading solution consists of WPM basal medium and 410.4g/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

(3) and (3) preserving the vitrified callus in liquid nitrogen.

In the method, the embryonic callus of the magnolia officinalis is mixed with a loading solution for loading treatment to obtain the loaded callus; the Loading solution consists of WPM basal medium, 184g/L glycerol and 136.8g/L sucrose. The preparation method of the magnolia bark embryonic callus preferably comprises the following steps of carrying out induction culture on magnolia bark seeds in an induction culture medium for 2 weeks; the induction culture medium consists of a WPM basic culture medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1g/L polyvinylpyrrolidone, 1g/L hydrolyzed casein, 40g/L sucrose and 3g/L plant gel, and the pH value is 5.8. The source of the magnolia officinalis seeds is not particularly limited, and preferably mature seeds are used as materials for callus induction. The induction culture of the present invention is preferably a dark culture, and the temperature of the dark culture is preferably 25 ℃. The preparation method of the induction medium is not particularly limited, and the components are mixed, sterilized at 121 ℃ and adjusted to pH 5.8. The composition of the WPM basal medium is shown in Table 1:

TABLE 1WPM basal Medium composition

Serial number	Compound (I)	Concentration mg/L
			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-inositol	100

When the mixing is carried out, the method preferably further comprises measuring a certain amount of the embryogenic callus, taking 0.25mL as an example, preferably adding 1mLWPM liquid culture medium into a sterilized 1.5mL centrifuge tube, and measuring the Magnolia cortex embryogenic callus with the volume of 0.25 mL. The WPM liquid culture medium is preferably prepared by proportioning the WPM basic culture medium according to the concentration shown in Table 1 and sterilizing.

When the magnolia officinalis embryogenic callus and the loading solution are mixed, the volume ratio of the magnolia officinalis embryogenic callus to the loading solution is preferably 1: 8. According to the invention, after the mixing, loading treatment is carried out, wherein the temperature of the loading treatment is preferably 25 ℃, and the time of the loading treatment is preferably 20 min. The preparation method of the Loading solution is not particularly limited in the invention, and the preparation method preferably comprises the steps of mixing the components, adjusting the pH value to 5.8, and carrying out high-pressure moist-heat sterilization at 121 ℃ for 15min and standing to normal temperature.

After obtaining the loaded callus, mixing the loaded callus with a vitrifying solution 2 solution (PVS2 solution) of ice for vitrifying to obtain a vitrified callus; the vitrification solution 2 solution (PVS2 solution) consists of WPM basic culture medium, 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide and 0.4mol/L sucrose. The present invention preferably further comprises, prior to said mixing, a loading solution (suction solution). The volume ratio of the loaded callus to the PVS2 solution is preferably 1:8 during the mixing process. The vitrification treatment is performed on ice, and the vitrification treatment time is preferably 30 min. The preparation method of the PVS2 solution is not particularly limited, but the components are preferably mixed, the pH value is adjusted to 5.8, and then the mixture is subjected to high-pressure moist heat sterilization at 121 ℃ for 15min and then is kept stand to room temperature. In the vitrification treatment, the PVS2 solution is pretreated, preferably, the PVS2 solution is transferred to ice for precooling treatment, so that ice PVS2 solution is obtained.

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 PVS2 solution 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 PVS2 solution in the thawed callus, mixing the thawed callus with Unloading solution (Unloading solution), and Unloading to obtain unloaded callus; the Unloading solution (Unloading solution) consists of WPM basal medium and 410.4g/L sucrose, and has pH 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. Selecting materials: the embryonic callus of the magnolia officinalis induced by the zygotic embryo of the mature seeds of the magnolia officinalis is cultured for 2 weeks under the dark condition of WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1g/LPVP (polyvinylpyrrolidone) +1g/L hydrolyzed Casein (CH) +40g/L sucrose +3g/L plant gel, pH 5.8 and 25 ℃.

2. Measuring 0.25mL of magnolia officinalis embryonic callus: 1mLWPM media was added to a sterilized 1.5mL centrifuge tube in a clean bench, with the scale level at the lowest level of the 1mL meniscus. Picking up the callus with good growth state by forceps, transferring into a 1.5mL centrifuge tube until the lowest part of the concave surface is equal to the scale of 1.25mL, and measuring the callus with volume of 0.25 mL.

3. Loading treatment: the pipette is equipped with 1mL of a clipped blue-gun head, 0.25mL of the weighed embryogenic callus is transferred to a 2mL cryopreservation tube, the WPM liquid is completely absorbed, and 2mL of loading solution is added for loading treatment for 20 minutes.

4. Vitrification treatment: at the end of the loading process, the loading solution was aspirated off with a pipette, 2mL of ice pre-chilled PVS2 solution was added and the mixture was treated on ice for 30 minutes.

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 treatment: after water bath, the outer surface of the frozen tube is wiped by 75% alcohol, the PVS2 solution is sucked up by a pipette in an ultra-clean workbench, 2mL of Unloading solution is added, and the tube is treated for 20 minutes at 25 ℃;

8. cleaning and restoring culture: and after the Unloading treatment is finished, sucking up the Unloading solution by using a pipette gun, adding 2mLWPM liquid culture medium for cleaning, and sucking up WPM liquid by using the pipette gun. After washing, 1mL of liquid LWPM was added to a 2mL vial. Installing a head with a cut opening by using a pipette, transferring the embryonic callus into a 9cm glass culture dish containing 3 pieces of filter paper, completely permeating the filtrate, and transferring to: WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1g/L PVP +1g/L CH +30g/L sucrose +3g/LPhytagel (plant gel), pH 5.8, and recovery culture was performed under dark condition at 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

At 1 month of recovery culture, embryogenic callus regenerated on filter paper was transferred to the following without filter paper: WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1g/L PVP +1g/L CH +30g/L sucrose +3g/L Phytagel (plant gel), pH 5.8, subcultured at 25 ℃ in the dark. The medium was changed every two weeks. After two subcultures, selecting embryogenic callus for two weeks, transferring to WPM culture medium for differentiation, generating somatic embryo after 1 month without callus, picking out somatic embryo, placing on WPM culture medium, transferring to tissue culture bottle for continuous culture after root and cotyledon grow out, transferring root system to seedling hole, and making into bagged seedling. The process of recovering the seedling is shown in figure 3, wherein a is the callus of magnolia officinalis embryo frozen and regenerated by liquid nitrogen; b is the somatic embryo of the regenerative embryogenic callus differentiation; c is a somatic embryo; d, tissue culture plantlets of magnolia officinalis; e is magnolia officinalis tissue culture bottle seedling; f is the magnolia officinalis plant transplanted into a seedling bag of 16 multiplied by 16cm, the seedling rate is 100 percent, and the survival rate is 100 percent.

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

1. A vitrification ultra-low temperature preservation method for magnolia officinalis embryonic callus is characterized by comprising the following steps: (1) mixing the cortex magnoliae officinalis embryonic callus with a loading solution for loading treatment to obtain loaded callus; the loading solution consists of WPM basal medium, 184g/L of glycerol and 136.8g/L of sucrose; the preparation method of the magnolia officinalis embryonic callus comprises the following steps of carrying out induction culture on magnolia officinalis seeds in an induction culture medium for 2 weeks; the induction culture medium consists of a WPM basic culture medium, 2 mg/L2, 4-D, 0.25 mg/L6-BA, 1g/L polyvinylpyrrolidone, 1g/L casein hydrolysate, 40g/L sucrose and 3g/L plant gel, and the pH value is 5.8; the induction culture is dark culture, and the temperature of the induction culture is 25 ℃; during mixing, the volume ratio of the magnolia officinalis embryonic callus to the loading solution is 1: 8;

(2) mixing the loaded callus with a vitrification solution 2 of ice for vitrification treatment to obtain vitrified callus; the vitrification solution 2 solution consists of a WPM basic culture medium, 300g/L of glycerol, 150g/L of ethylene glycol, 150g/L of dimethyl sulfoxide and 0.4mol/L of cane sugar; before the mixing, the method also comprises the step of sucking and discarding the loading solution; during the mixing, the volume ratio of the loading callus to the vitrification solution 2 is 1: 8; the vitrification treatment is carried out on ice, and the vitrification treatment time is 30 min;

(3) and (3) preserving the vitrified callus in liquid nitrogen.

2. The method of claim 1, further comprising restoring the preserved callus after said preserving in step (3).

3. The method of claim 2, wherein the holding time is at least 24 hours.

4. The method of claim 2, wherein the recovering method comprises the steps of: (a) unfreezing the preserved callus in a water bath at 40 ℃;

5. The method of claim 4, 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).