CN114617064B - Preservation method of magnolia sieboldii germplasm resource - Google Patents
Preservation method of magnolia sieboldii germplasm resource Download PDFInfo
- Publication number
- CN114617064B CN114617064B CN202210291018.6A CN202210291018A CN114617064B CN 114617064 B CN114617064 B CN 114617064B CN 202210291018 A CN202210291018 A CN 202210291018A CN 114617064 B CN114617064 B CN 114617064B
- Authority
- CN
- China
- Prior art keywords
- callus
- solution
- preservation
- magnolia
- wpm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- A01H4/002—Culture media for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of quality preservation of magnolia denudata, in particular to a method for preserving the quality resources of the magnolia denudata. The preservation method of the magnolia sieboldii germplasm resource utilizes the embryonic callus to carry out loading treatment in a proper loading solution and vitrification treatment in a proper vitrification solution, thereby completing ultralow temperature preservation. Compared with the existing in-situ establishment of a protection area for storage and ex-situ storage, the method provided by the invention has the advantages of permanent storage, no influence of natural environment change, high storage efficiency, high genetic stability and convenience in management. In addition, the preserved vitrified callus can be quickly proliferated and differentiated after regeneration, and differentiated somatic embryos are directly developed into plants, and the transformation rate is high without rooting culture. The regeneration rate of the ultra-low temperature preservation is 90%, and the ultra-low temperature preservation can be carried out on the magnolia sieboldii germplasm resources for a long time.
Description
Technical Field
The invention relates to the technical field of quality preservation of magnolia denudata, in particular to a method for preserving the quality resources of the magnolia denudata.
Background
Magnolia red flower (Magnolia delavayi) is a variety of Magnolia red of the Magnolia genus of Magnoliaceae. The tree shape is beautiful, the leaf shape is beautiful, the flower is large and gorgeous and red, the flower has fragrance and certain anti-fouling effect, and the tree is an excellent tree species for garden landscape and urban greening. At present, yunnan province has taken the same as the country tree species as the famous book of urban greening tree species of Yunnan province, and is applied to urban greening as street trees and ornamental trees in Yunnan, yunnan West, yunnan northwest, yunnan southeast and the like.
At present, the breeding mode of the magnolia sieboldii is mainly seed breeding. The number of magnolia sieboldii population is less, and the seeds produced by the magnolia sieboldii population are less. In addition, the magnolia denudata is female, male and heteromature, so that pollination of insects is hindered, the setting rate is low in a natural state, fructification is easily influenced by climatic conditions and a habitat, and certain difficulty exists in obtaining a large number of seeds for plant propagation. Due to the change of natural conditions, the resources and distribution are sharply reduced, and the wild plants are listed as national secondary protection wild plants. Therefore, the preservation of the species resource of the magnolia sieboldii becomes the key for the sustainable utilization of the magnolia sieboldii. At present, germplasm resources of magnolia sieboldii are mostly preserved by establishing a protection area and introducing and preserving in an ex-situ manner.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preserving a magnolia sieboldii germplasm resource. The method provided by the invention is simple, convenient and feasible, has high stability, high efficiency and reliability, can be rapidly differentiated into somatic embryos of magnolia delavayi after being stored by utilizing the differentiation capability of embryonic callus, and further develops into complete plants, and the plants have good growth state after being transplanted.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preservation method of magnolia sieboldii germplasm resources, which comprises the following steps:
(1) Mixing the embryonic callus of the magnolia sieboldii with a loading solution for loading treatment to obtain loaded callus; the loading solution comprises: WPM basic culture medium, glycerol 184g/L and sucrose 136.8g/L;
(2) Mixing the loaded callus with a vitrification solution for vitrification treatment to obtain vitrified callus; the vitrification solution comprises 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 the embryonic callus of magnolia sieboldii in the step (1) comprises the following steps: the method comprises the following steps of carrying out induction culture on magnolia delavayi seeds in an induction culture medium for 4 months, changing the culture medium every 1 month, wherein the induction culture medium takes WPM as a basic culture medium, and further comprises the following components: 2,4-D2 mg/L, 6-BA0.25mg/L, polyvinylpyrrolidone 1g/L, hydrolyzed casein 1g/L, sucrose 40g/L and plant gel 3g/L, wherein the pH value of the induction medium 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 sieboldii embryogenic callus to the loading solution during the mixing in the step (1) is 1:8.
Preferably, the volume ratio of the loaded callus to the vitrification solution in the mixing of the step (2) is 1:8.
Preferably, the vitrification treatment temperature in the step (2) is 0-4 ℃, and the vitrification treatment time is 30min.
Preferably, the storage time in step (3) is at least 24h.
Preferably, after the preservation in step (3), restoring the preserved callus is further included.
Preferably, the recovery method comprises the following steps:
(a) Unfreezing the preserved callus in a water bath at 40 ℃;
(b) Mixing the thawed callus with an unloading solution, and then carrying out unloading treatment to obtain unloaded callus; the unloading solution comprises WPM basic culture medium and cane sugar 410.4g/L, and the pH value of the unloading solution is 5.8;
(c) Transferring the unloaded callus 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 broth, mixing the washed unloaded callus with the WPM broth, and finally removing the WPM broth.
Has the beneficial effects that:
the invention provides a method for storing a magnolia sieboldii germplasm resource, which comprises the following steps: (1) Mixing the embryonic callus of the magnolia sieboldii with a loading solution for loading treatment to obtain loaded callus; the loading solution comprises: WPM basic culture medium, glycerol 184g/L and sucrose 136.8g/L; (2) Mixing the loaded callus with a vitrification solution for vitrification treatment to obtain vitrified callus; the vitrification solution comprises 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. The preservation method of the magnolia sieboldii germplasm resource is based on the characteristics that the proliferation rate of the regenerated magnolia sieboldii 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, the embryonic callus is loaded in a proper loading solution, and vitrification treatment is carried out in a proper vitrification solution, so that the ultralow temperature preservation is completed.
Compared with the existing in-situ establishment of protected area storage and ex-situ storage, the method provided by the invention has the advantages of permanent storage, no influence of natural environment change, high storage efficiency, high genetic stability and convenience in management.
In addition, the preserved vitrified callus can be quickly proliferated and differentiated after regeneration, and differentiated somatic embryos can be directly developed into plants, and the transformation rate is high, so that rooting culture is not needed. The regeneration rate of the ultra-low temperature preservation is 90%, and the ultra-low temperature preservation can be carried out on the magnolia sieboldii germplasm resources for a long time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 shows the survival rate of embryogenic callus of Magnolia liliiflora at ultra-low temperature;
FIG. 2 is a recovery process of ultra-low temperature preservation of embryonic callus of Magnolia liliiflora; wherein a is a raw material for two weeks of subculture at ultra-low temperature; b, recovering and culturing the embryonic callus of the magnolia sieboldii for 48 hours after thawing; c is the ultra-low temperature regenerated magnolia sieboldii embryonic callus;
FIG. 3 shows the differentiation and seedling process of the regenerated embryonic callus, wherein a is the regenerated embryonic callus of Magnolia sieboldii frozen in liquid nitrogen, and the lower right-angle scale in a is 1mm; b is a regenerated embryogenic callus differentiated somatic embryo, and the middle-right lower angle scale of b is 500 mu m; c is a somatic embryo, and the middle right lower angle scale of c is 200 mu m; d, tissue culture seedlings of the magnolia sieboldii; e is tissue culture bottle seedling of Magnolia liliiflora; f is a magnolia sieboldii plant transplanted into a seedling bag with the length of 16 multiplied by 16 cm; g is a 1-year-old bag containing flos Carthami and flos Magnoliae;
FIG. 4 shows the survival rate of embryogenic callus of Michelia sphaerica at ultra-low temperature.
Detailed Description
The invention provides a method for storing a magnolia sieboldii germplasm resource, which comprises the following steps:
(1) Mixing the embryonic callus of the magnolia sieboldii with a loading solution for loading treatment to obtain loaded callus; the loading solution comprises: WPM basic culture medium, glycerol 184g/L and sucrose 136.8g/L;
(2) Mixing the loaded callus with a vitrification solution for vitrification treatment to obtain vitrified callus; the vitrification solution comprises a WPM basic culture medium, 300g/L of glycerol, 150g/L of glycol, 150g/L of dimethyl sulfoxide and 0.4mol/L of cane sugar;
(3) And (3) preserving the vitrified callus in liquid nitrogen.
The invention mixes the magnolia sieboldii embryonic callus with the loading solution for loading treatment to obtain the loaded callus. In the present invention, the method for preparing the embryonic callus of magnolia sieboldii preferably comprises: inducing and culturing the magnolia sieboldii seeds in an induction culture medium for 4 months, and replacing the culture medium every 1 month; more preferably, it comprises: disinfecting the magnolia delavayi seeds, stripping zygotic embryos, carrying out induced culture on the zygotic embryos in an induction culture medium for 4 months, and changing the culture medium every 1 month; in the present invention, the source of the magnolia delavayi Franch seed is not particularly limited, but preferably callus induction is performed using mature seeds as a material.
In the invention, the induction medium takes WPM as a basic medium, and preferably further comprises the following components: 2,4-D2 mg/L, 6-BA0.25mg/L, polyvinylpyrrolidone 1g/L, hydrolyzed casein 1g/L, sucrose 40g/L and plant gel 3g/L, wherein the pH value of the induction medium is preferably 5.8; the induction culture is preferably a dark culture, and the temperature of the induction culture is preferably 25 ℃. The method for preparing the induction medium is not particularly limited, and the method is preferably: mixing the above materials, sterilizing at 121 deg.C, and adjusting pH to 5.8. In the present invention, the WPM medium preferably includes the following components: NH (NH) 4 N0 3 400mg/L、Ca(NO 3 ) 2· 4H 2 O 556mg/L、K 2 SO 4 990 mg/L、CaCl 2 ·2H 2 O 96mg/L、KH 2 PO 4 170mg/L、H 3 BO 3 6.2mg/L、Na 2 MoO 4 ·2H 2 O 0.25mg/L、MgSO 4 ·7H 2 O 370mg/L、MnSO 4 ·H 2 O 22.4mg/L、ZnSO 4 ·7H 2 O 8.6mg/L、CuSO 4 ·5H 2 O 0.25mg/L、FeSO 4 ·7H 2 O 27.8mg/L、Na 2 37.3mg/L EDTA, 100mg/L inositol, 11.0mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, 2.0mg/L glycine, 30g/L sucrose and 3g/L plant gel; the WPM medium preferably has a pH of 5.7.
In the present invention, the volume ratio of the magnolia delavayi embryogenic callus to the loading solution is preferably 1:8. According to the invention, after the mixing, the loading treatment is carried out, the temperature of the loading treatment is preferably 25 ℃, and the time of the loading treatment is preferably 20min.
After the loaded callus is obtained, the loaded callus is mixed with a vitrification solution for vitrification treatment to obtain the vitrified callus. The present invention preferably further comprises, prior to said mixing, removing the loading solution from the loaded callus; the means of removal preferably comprises suction. In the vitrification treatment of the present invention, it is preferable that the vitrification solution is pretreated, including: and (4) transferring the vitrified solution to ice for precooling treatment to obtain the ice vitrified solution. The volume ratio of the loaded callus to the vitrification solution in the mixing of the present invention is preferably 1:8. The temperature of the vitrification treatment is 0-4 ℃, the vitrification treatment is preferably carried out on ice, and the vitrification treatment time is preferably 30min. The preparation method of the vitrification solution is not particularly limited in the present invention, and the above components are preferably mixed, adjusted to pH 5.8, sterilized by moist heat at 121 ℃ for 15min, and then left to stand to room temperature.
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 vitrified solution is placed in a cryopreservation tube, fixed on a bracket of the cryopreservation 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 24h.
In the present invention, it is preferable that the method further comprises restoring the callus after the storage of the vitrified callus.
In the present invention, the method for recovering preferably comprises the following steps:
(a) Unfreezing the preserved callus in a water bath at 40 ℃;
(b) Mixing the thawed callus with an unloading solution, and then carrying out unloading treatment to obtain unloaded callus; the unloading solution comprises WPM basic culture medium and cane sugar 410.4g/L, and the pH value of the unloading solution is 5.8;
(c) Transferring the unloaded callus into the induction medium for recovery culture.
The invention unfreezes the preserved callus in water bath at 40 ℃. In the present invention, the time for thawing is preferably 2min.
After the thawed callus is obtained, the thawed callus is mixed with the unloading solution and then is unloaded to obtain the unloaded callus. In the present invention, before mixing the thawed callus with the unloading solution, it is preferable to further comprise removing the vitrification solution in the thawed callus; the means of removal preferably comprises suction.
Before the unloading treatment, the invention preferably also comprises the step of disinfecting the surface of the cryopreservation tube after the water bath, wherein the disinfection is preferably performed by wiping with alcohol with the volume fraction of 75%, and then the cryopreservation tube is placed in an ultra-clean workbench for the unloading treatment. The amount of the unloading solution according to the invention is preferably the same as the amount of the loading solution. The unloading treatment according to the invention is preferably carried out at 25 ℃ for 20min.
After obtaining the unloaded callus, the invention transfers the unloaded callus to the induction medium for recovery culture.
After the unloading of the processing result and before the transferring, the present invention preferably further comprises: and (3) cleaning the unloaded callus by using a WPM liquid culture medium, mixing the cleaned 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 embryonic callus of two weeks after two subcultures, transfers the embryonic 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, after 2-3 true leaves grow, transfers more than 3 fibrous roots to seedling raising holes, finally makes bag seedling.
In order to further illustrate the present invention, the method for preserving magnolia sieboldii germplasm resources provided by the present invention is described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
A method for storing species resources of Magnolia sieboldii comprises the following steps:
1. inducing and culturing zygotic embryos of mature seeds of magnolia delavayi Franch in an induction culture medium (culturing at 25 ℃ under dark condition) for 4 months continuously, and changing the culture medium every 1 month to obtain embryonic callus of magnolia delavayi Franch; the induction culture medium takes WPM as a basic culture medium and only contains the following components: 2,4-D2 mg/L, 6-BA0.25mg/L, polyvinylpyrrolidone 1g/L, hydrolyzed casein 1g/L, sucrose 40g/L and plant gel 3g/L, wherein the pH value of the induction medium is 5.8.
2. 1mLWPM volume medium was added to sterilized 1.5mL centrifuge tubes in a clean bench. Picking the embryonic callus of the magnolia sieboldii with good growth state by using forceps, transferring the embryonic callus into a 1.5mL centrifuge tube until the lowest part of the concave liquid surface is leveled with a scale of 1.25mL, and measuring the embryonic callus of the magnolia sieboldii with the volume of 0.25 mL.
3. Loading treatment: loading a 1mL blue gun head with a cut opening on a pipette, transferring 0.25mL embryonic callus measured into a 2mL freezing tube, completely sucking WPM liquid, adding 2mL loading solution, and loading for 20min; the loading solution consists of WPM basal medium, 184g/L of glycerol and 136.8g/L of sucrose.
4. Vitrification treatment: after the loading treatment is finished, completely absorbing the loading solution by using a pipette, adding 2mL of a vitrified solution precooled on ice, and treating for 30min on the ice; the vitrification solution consists of 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.
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 quickly placing the freezing tube bracket into a liquid nitrogen tank for preservation 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 for more than 24 hours in liquid nitrogen 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 2min.
7. Unloading treatment: freezing the tube after water bath, wiping the outer surface of the tube with 75% alcohol, sucking out the vitrification solution in an ultra-clean workbench by using a liquid transfer gun, adding 2mL unloading solution, and treating for 20min at 25 ℃; the unloading solution consists of WPM basic culture medium and cane sugar 410.4g/L, and the pH value of the unloading solution is 5.8.
8. Cleaning and restoring culture: and after the unloading treatment is finished, completely sucking the unloading solution by using a pipette gun, adding 2mLWPM liquid culture medium for cleaning, and completely sucking WPM liquid by using the pipette gun. After washing, 1mL of liquid was added to a 2mL vial. And (3) installing a cut gun head by using a liquid transfer gun, transferring the embryonic callus into a 9cm glass culture dish containing 3 pieces of filter paper, completely permeating the filtrate, transferring the filtrate into the induction culture medium obtained in the step 1, and performing recovery culture at 25 ℃ in the dark.
9. Identification of survival rate and recovery ability and selection of optimal vitrification solution treatment time
And (3) dyeing with a Fluorescein Diacetate (FDA) live cell fluorescent dye and a Propidium Iodide (PI) dead cell fluorescent dye, and carrying out survival rate detection on the embryonic callus of the magnolia sieboldii for 48h by using a laser confocal microscope, wherein the survival is determined when the fluorescence value is present, and the death is determined when the fluorescence value is absent. After the thawed embryonic callus is restored and cultured for two weeks, the state of the embryonic callus is directly observed. The yellow and proliferating embryogenic callus is alive, all white and non-proliferating is dead. According to the survival condition of each cryopreserved tubular embryogenic callus, the regeneration rate is calculated. The FDA staining was consistent with the directly observed statistics. And the recovery culture time is 48h, which is the most accurate time point for determining the survival rate of the embryonic callus of the magnolia sieboldii.
Thereby obtaining the most accurate time point for measuring the survival rate of the embryonic callus of the magnolia sieboldii in 48 hours.
The embryonic callus after two weeks of freezing of 30 tubes is subjected to recovery culture, 5 frozen tubes are subjected to each experiment, 6 experiments are carried out, the number of the tubes subjected to 2 consecutive experiments is recorded as 1 repetition, 3 repetitions are counted, and the results are shown in figure 1, wherein 10 tubes are recovered at 1, 2 and 3 times, and the average survival rate after recovery is 90%.
And then processing the vitrification solution for 0min,5min, 10min,30min, 50min, 70min and 90min, preserving at ultralow temperature, recovering fluorescence and fluorescence intensity of 48h of culture, and recovering 48h of result, wherein the vitrification solution is processed for 30min 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 restoration culture of the Magnolia sieboldii embryogenic callus after thawing for 48h, and c is the ultra-low temperature regeneration Magnolia sieboldii embryogenic callus) was combined to obtain 30min, which is the most vitrification treatment time of the Magnolia sieboldii embryogenic callus.
10. Subculture, differentiation and seedling formation of regenerated embryonic callus
After 1 month of recovery culture, the embryogenic callus regenerated on the filter paper was transferred to an induction medium without filter paper (the induction medium is similar to that in step 1 except that the concentration of sucrose was 30 g/L), and subcultured in the dark at 25 ℃. The medium was changed every two weeks. After two subcultures, selecting embryonic callus for two weeks, transferring the embryonic callus to a WPM culture medium to be differentiated, generating somatic embryos after 1 month without the callus, picking out the somatic embryos, placing the somatic embryos on the WPM culture medium, transferring the somatic embryos to a tissue culture bottle for continuous culture after roots and cotyledons begin to grow out, sending root systems to seedling raising holes, and finally making bag seedlings. The process of recovering the seedling is shown in figure 3, wherein a is the embryonic callus of the red magnolia denudata which is 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 seedlings of the magnolia sieboldii; e is tissue culture bottle seedling of Magnolia liliiflora; f is a magnolia sieboldii plant transplanted into a seedling bag with the length of 16 multiplied by 16cm, and g is a magnolia sieboldii bag grown for 1 year.
As can be seen from the fig. 3 and fig. 1, the embryonic cells of the magnolia denudata can be safely and effectively preserved for a long time by the vitrification cryopreservation technology, and the survival rate reaches 90 percent; embryonic cells after ultralow temperature preservation can be rapidly differentiated into magnolia sieboldii plants; the differentiated magnolia delavayi can be transplanted to survive under natural conditions, the survival rate of the transplantation reaches 90%, and the later growth is good.
Comparative example 1
A method similar to example 1, except that the zygotic embryos of the mature seeds of magnolia delavayi are replaced with the zygotic embryos of the mature seeds of michelia sphaera. And the embryonic callus after being frozen for two weeks is subjected to recovery culture by the same method, the result is shown in figure 4, and the average survival rate after recovery is 36.7%.
As can be seen from the example 1 and the comparative example 1, the method provided by the invention can be used for carrying out ultralow-temperature preservation on the magnolia sieboldii germplasm resource for a long time, but has poor preservation effect on michelia figo of Magnoliaceae.
In conclusion, compared with the existing in-situ establishment of protected area storage and ex-situ storage, the method provided by the invention has the advantages of permanent storage, no influence of natural environment change, high storage efficiency, high genetic stability and convenience in management. In addition, the preserved vitrified callus can be quickly proliferated and differentiated after regeneration, and differentiated somatic embryos can be directly developed into plants, and the transformation rate is high, so that rooting culture is not needed. The regeneration rate of the ultra-low temperature preservation is 90%, and the ultra-low temperature preservation can be carried out on the magnolia sieboldii germplasm resources for a long time.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. A method for storing seed quality resources of magnolia sieboldii is characterized by comprising the following steps:
(1) Mixing the embryonic callus of the magnolia sieboldii with a loading solution for loading treatment to obtain loaded callus; the loading solution comprises: WPM basic culture medium, glycerol 184g/L and sucrose 136.8g/L;
the preparation method of the embryonic callus of the magnolia sieboldii comprises the following steps: carrying out induction culture on the magnolia delavayi seeds in an induction culture medium for 4 months, and changing the culture medium every 1 month; the induction culture medium takes WPM as a basic culture medium and only contains the following components: 2,4-D2 mg/L, 6-BA0.25mg/L, polyvinylpyrrolidone 1g/L, hydrolyzed casein 1g/L, sucrose 40g/L and plant gel 3g/L, wherein the pH value of the induction medium is 5.8;
the volume ratio of the magnolia sieboldii embryonic callus to the loading solution is 1:8;
(2) Mixing the loaded callus with a vitrification solution for vitrification treatment to obtain vitrified callus; the vitrification solution comprises 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; the volume ratio of the loaded callus to the vitrification solution is 1:8;
(3) Placing the vitrified callus in liquid nitrogen for preservation; after the preservation, restoring the preserved callus;
the recovery method comprises the following steps:
(a) Unfreezing the preserved callus in a water bath at 40 ℃;
(b) Mixing the thawed callus with an unloading solution, and then carrying out unloading treatment to obtain unloaded callus; the unloading solution comprises WPM basic culture medium and cane sugar 410.4g/L, and the pH value of the unloading solution is 5.8;
(c) Transferring the unloaded callus to the induction medium for recovery culture.
2. The preservation method according to claim 1, wherein the induction culture is a dark culture and the temperature of the induction culture is 25 ℃.
3. The preservation method according to claim 1, wherein the temperature of the vitrification treatment in the step (2) is 0 to 4 ℃, and the time of the vitrification treatment is 30min.
4. The preservation method according to claim 1, wherein the preservation time of step (3) is at least 24h.
5. The preservation method according to claim 1, further comprising washing the unloaded callus with WPM broth, mixing the washed unloaded callus with WPM broth, and removing the WPM broth before the transferring in step (c).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210291018.6A CN114617064B (en) | 2022-03-23 | 2022-03-23 | Preservation method of magnolia sieboldii germplasm resource |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210291018.6A CN114617064B (en) | 2022-03-23 | 2022-03-23 | Preservation method of magnolia sieboldii germplasm resource |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114617064A CN114617064A (en) | 2022-06-14 |
| CN114617064B true CN114617064B (en) | 2022-12-20 |
Family
ID=81903964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210291018.6A Active CN114617064B (en) | 2022-03-23 | 2022-03-23 | Preservation method of magnolia sieboldii germplasm resource |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114617064B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111387176B (en) * | 2020-04-24 | 2021-07-30 | 中国科学院昆明植物研究所 | Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus |
| CN111972402A (en) * | 2020-09-04 | 2020-11-24 | 贵州大学 | Vitrification cryopreservation and thawing method for agate red cherry embryogenic callus |
-
2022
- 2022-03-23 CN CN202210291018.6A patent/CN114617064B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114617064A (en) | 2022-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100889342B1 (en) | Propagation method of liriodendron tulipifera using somatic embryogenesis technique | |
| CN102648698A (en) | Pyrus stem tip tissue culture rapid propagation method | |
| CN109757373A (en) | A kind of Jing Banxia quick breeding method for tissue culture | |
| CN104381131B (en) | A kind of Pinus tabuliformis somatic embryo occurs and plant regeneration method | |
| CN106665357A (en) | Method for establishing lycoris regeneration system | |
| CN107155898A (en) | A kind of dendrobium candidum carries out expanding numerous method using stem section thin slice | |
| CN107278891B (en) | A kind of apricot plum quick breeding method for tissue culture | |
| CN103609453B (en) | A kind of construction method of tea tree vitro Regeneration System | |
| CN102613087B (en) | Method for culturing and breeding Correa carmen by using biological tissue | |
| CN111387176B (en) | Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus | |
| CN101983555A (en) | Method for inducing indirect somatic embryogenesis of chrysanthemum | |
| CN104012406B (en) | The in-vitro regeneration method of sweet cherry variety red pearl in evening | |
| KR101531308B1 (en) | Method of propagating Paulownia coreana using tissue culture | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN114617064B (en) | Preservation method of magnolia sieboldii germplasm resource | |
| CN105010123B (en) | The method and culture medium of strawberry distant hybrid are obtained by rescue isolated culture | |
| KR101106953B1 (en) | Proliferation method of Pinus densiflora using somatic embryogenesis | |
| CN108849500A (en) | The culture medium and method of a kind of rescue of lotus embryo and the development of offspring's fast-growth | |
| CN108271693A (en) | One kind posting tree orchid regenerating system quickly foundation and store method | |
| CN107484665A (en) | A kind of method using black fruit fructus lycii resting shoot seedling | |
| Behera et al. | Effect of plant growth regulator on in-vitro micropropagation of Bitter Yam (Dioscorea hispida Dennst.) | |
| KR100302206B1 (en) | In-flight mass production and forge transplanting method | |
| CN100387113C (en) | Tissue cultivation quick breeding method for red leaf Chinese toon | |
| CN103053429A (en) | Method for regenerating semen pharbitidis in vitro embryonic axis plant | |
| Saborío et al. | Development of a clean-planting-material production system on tropical root and tuber crops, using in vitro propagated plants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |