NL2031306B1 - Method for vitrification and ultra-low-temperature preservation of embryogenic callus of magnolia officinalis - Google Patents
Method for vitrification and ultra-low-temperature preservation of embryogenic callus of magnolia officinalis Download PDFInfo
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- NL2031306B1 NL2031306B1 NL2031306A NL2031306A NL2031306B1 NL 2031306 B1 NL2031306 B1 NL 2031306B1 NL 2031306 A NL2031306 A NL 2031306A NL 2031306 A NL2031306 A NL 2031306A NL 2031306 B1 NL2031306 B1 NL 2031306B1
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- callus
- magnolia officinalis
- vitrification
- embryogenic callus
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 86
- 230000000408 embryogenic effect Effects 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000004017 vitrification Methods 0.000 title claims abstract description 31
- 238000004321 preservation Methods 0.000 title claims abstract description 27
- 241001673966 Magnolia officinalis Species 0.000 title claims description 51
- 239000001963 growth medium Substances 0.000 claims description 33
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 241000196324 Embryophyta Species 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 239000005720 sucrose Substances 0.000 claims description 15
- 230000006698 induction Effects 0.000 claims description 14
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 229920002148 Gellan gum Polymers 0.000 claims description 8
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 7
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 7
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 7
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims 1
- 235000021240 caseins Nutrition 0.000 claims 1
- 241000218378 Magnolia Species 0.000 abstract 2
- 238000011084 recovery Methods 0.000 description 16
- 210000002257 embryonic structure Anatomy 0.000 description 8
- 230000000392 somatic effect Effects 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- 108010079058 casein hydrolysate Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 3
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011066 ex-situ storage Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Agronomy & Crop Science (AREA)
- Physiology (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present disclosure provides a method for vitrification and ultra-low-temperature preservation of an embryogenic callus of Magnolia ojficinalis, and belongs to the technical 5 field of preservation of germplasms of Magnolia ojficinalz‘s.
Description
METHOD FOR VITRIFICATION AND ULTRA-LOW-TEMPERATURE
PRESERVATION OF EMBRYOGENIC CALLUS OF MAGNOLIA OFFICINALIS
[OI] The present disclosure belongs to the technical field of preservation of germplasms of Magnolia officinalis, and specifically relates to a method for vitrification and ultra-low- temperature preservation of an embryogenic callus of Magnolia officinalis.
[02] Magnolia officinalis has not only an important medicinal value, but also a high ornamental value. As the bark of the Magnolia officinalis can be prepared into medicines, over-cutting of the Magnolia officinalis 1s caused, resulting in rapid decrease of resources and distribution of the Magnolia officinalis. At present, the Magnolia officinalis has been listed as a national second-level protected wild plant. Therefore, preservation of germplasm resources of the Magnolia officinalis has become the key to achieve sustainable utilization of the Magnolia officinalis. At present, the germplasm resources of the Magnolia officinalis are mainly preserved by establishing a protected area and conducting artificial planting.
[03] Inview of this situation, an objective of the present disclosure 1s to provide a method for vitrification and ultra-low-temperature preservation of an embryogenic callus of
Magnolia officinalis. The method is simple and easy to implement and has high stability, high efficiency and reliability. After being preserved, the embryogenic callus with a differentiation ability can be rapidly differentiated into a large number of somatic embryos of the Magnolia officinalis and further developed into a complete plant. In addition, the plant grows well after being transplanted.
[04] To achieve the objective of the present disclosure, the present disclosure provides the following technical solutions.
[05] The present disclosure provides a method for vitrification and ultra-low-temperature preservation of an embryogenic callus of Magnolia officinalis. The method includes the following steps: (1) mixing the embryogenic callus of the Magnolia officinalis and a loading solution for loading to obtain a loaded callus, where, the loading solution includes a WPM basic culture medium, 184 g/L of glycerol and 136.8 g/L of sucrose;
[06] (2) mixing the loaded callus and an icy plant vitrification solution II for vitrification to obtain a vitrified callus, where, the plant vitrification solution II includes a WPM basic culture medium, 300 g/L of glycerol, 150 g/L of ethylene glycol, 150 g/L of dimethyl sulfoxide and 0.4 mol/L of sucrose; and
[07] (3) preserving the vitrified callus in liquid nitrogen.
[08] Preferably, a preparation method of the embryogenic callus of the Magnolia officinalis in step (1) includes conducting induction culture on Magnolia officinalis seeds in an induction culture medium for 2 weeks. The induction culture medium includes a WPM basic culture medium, 2 mg/L of 2,4-D, 0.25 mg/L of 6-BA, 1 g/L of polyvinylpyrrolidone, 1 g/L of a casein hydrolysate, 40 g/L of sucrose and 3 g/L of phytagel, and the pH is 5.8.
[09] Compared with the prior art, the present disclosure has the following beneficial effects. According to the method for vitrification and ultra-low-temperature preservation of an embryogenic callus of Magnolia officinalis in the present disclosure, the regenerated embryogenic callus of the Magnolia officinalis has the characteristics of being high in proliferation rate and capable of being proliferated and differentiated for a long time, differentiated into the somatic embryos with high efficiency and transformed into the plant with high efficiency. The embryogenic callus is subjected to the loading and the plant vitrification, and then the ultra-low-temperature preservation is completed. The loading and the plant vitrification are conducted to reduce the content of water in cells and improve the permeability of the cells. In this way, when the cells are put in the liquid nitrogen, icing in the cells can be reduced, and the integrity of cell membranes is further protected.
[10] Compared with existing methods of establishing a protected area for in-situ preservation and conducting ex-situ preservation, the method has the advantages of permanent preservation without being affected by changes of the natural environment, high preservation efficiency and convenient management. Compared with an existing tissue culture preservation method, the method has the advantages that the genetic stability is high, the preservation efficiency is high, the operation is fast and simple, the regenerated callus can be rapidly proliferated and differentiated, and the differentiated somatic embryos are directly develop into the plant in a high transformation rate without rooting culture. In the present disclosure, a regeneration rate during the ultra-low-temperature preservation is 100%, and germplasm resources of the Magnolia officinalis can be preserved at an ultra-low temperature for a long time.
[11] FIG. I shows a survival rate of an embryogenic callus of Magnolia officinalis during ultra-low-temperature preservation;
[12] FIG. 2 shows a recovery process of an embryogenic callus of Magnolia officinalis during ultra-low-temperature preservation; and
[13] FIG. 3 shows differentiation and seedling processes of a regenerated embryogenic callus.
[14] The present disclosure provides a method for vitrification and ultra-low-temperature preservation of an embryogenic callus of Magnolia officinalis. The method includes the following steps: (1) the embryogenic callus of the Magnolia officinalis and a loading solution are mixed for loading to obtain a loaded callus, where, the loading solution includes a WPM basic culture medium, 184 g/L of glycerol and 136.8 g/L of sucrose;
[5] (2) the loaded callus and an icy plant vitrification solution II are mixed for vitrification to obtain a vitrified callus, where, the plant vitrification solution II includes a
WPM basic culture medium, 300 g/L of glycerol, 150 g/L of ethylene glycol, 150 g/L of dimethyl sulfoxide and 0.4 mol/L of sucrose; and
[16] (3) the vitrified callus is preserved in liquid nitrogen.
[17] According to the method of the present disclosure, the embryogenic callus of the
Magnolia officinalis and the loading solution are mixed for the loading to obtain the loaded callus. The loading solution includes the WPM basic culture medium, 184 g/L of the glycerol and 136.8 g/L of the sucrose. In the present disclosure, a preparation method of the embryogenic callus of the Magnolia officinalis preferably includes that induction culture is conducted on Magnolia officinalis seeds in an induction culture medium for 2 weeks. The induction culture medium includes a WPM basic culture medium, 2 mg/L of 2,4-D, 0.25 mg/L of 6-BA, 1 g/L of polyvinylpyrrolidone, 1 g/L of a casein hydrolysate, 40 g/L of sucrose and 3 g/L of phytagel, and the pH is 5.8. In the present disclosure, sources of the
Magnolia officinalis seeds are not particularly limited, and mature seeds are preferably used as a material for callus induction. In the present disclosure, the induction culture is preferably dark culture, and the dark culture is preferably conducted at a temperature of 25°C. In the present disclosure, a preparation method of the induction culture medium is not particularly limited. The method includes that the components above are mixed, sterilization is conducted at 121°C, and then the pH is adjusted to 5.8. In the present disclosure, compositions of the WPM basic culture medium are shown in Table 1.
[18] Table 1 Compositions of the WPM basic culture medium
[19]
Tee pw co mw wo
TO Jesosmo jos momo ve
[20] In the present disclosure, when the embryogenic callus and the loading solution are mixed, the method preferably further includes that a certain amount of the embryogenic callus is measured. With 0.25 mL of the embryogenic callus as an example, 1 mL of a WPM liquid culture medium is preferably added into 1.5 mL of a sterilized centrifuge tube, and then the embryogenic callus of the Magnolia officinalis with a volume of 0.25 mL is measured. In the present disclosure, the WPM liquid culture medium 1s preferably obtained by preparing the WPM basic culture medium according to a concentration shown in Table 1 and then conducting sterilization and disinfection.
[21] In the present disclosure, the embryogenic callus of the Afagnolia officinalis and the loading solution are preferably mixed at a volume ratio of 1:8. In the present disclosure, the loading is conducted after the embryogenic callus and the loading solution are mixed, and the loading is preferably conducted at a temperature of 25°C for 20 min. In the present disclosure, a preparation method of the loading solution is not particularly limited. The method preferably includes that the components above are mixed, the pH is adjusted to 5.8,
moist heat sterilization is conducted by using an autoclave at 121°C for 15 min, and then the loading solution is placed at room temperature.
[22] In the present disclosure, the obtained loaded callus and the icy plant vitrification solution II (PVS2 solution) are mixed for the vitrification to obtain the vitrified callus. The 5 PVS2 solution includes the WPM basic culture medium, 300 g/L of the glycerol, 150 g/L of the ethylene glycol, 150 g/L of the dimethyl sulfoxide and 0.4 mol/L of the sucrose. In the present disclosure, before the loaded callus and the PVS2 solution are mixed, the method preferably further includes that the loading solution is removed by suction. In the present disclosure, the loaded callus and the PVS2 solution are preferably mixed at a volume ratio of 1:8. In the present disclosure, the vitrification is conducted on ice, and the vitrification is preferably conducted for 30 min. In the present disclosure, a preparation method of the PVS2 solution is not particularly limited. The method preferably includes that the components above are mixed, the pH is adjusted to 5.8, moist heat sterilization is conducted by using an autoclave at 121°C for 15 min, and then the PV S2 solution is placed at room temperature.
Inthe present disclosure, when the vitrification is conducted, the PVS2 solution is pretreated.
The PVS2 solution is preferably pre-cooled on ice to obtain an icy PVS2 solution.
[23] In the present disclosure, the obtained vitrified callus is preserved in the liquid nitrogen. In the present disclosure, when the vitrified callus is preserved, a mixture of the vitrified callus and the PVS2 solution is preferably placed in a cryogenic tube, fixed to a cryogenic tube support and then preserved in a liquid nitrogen tank.
[24] Inthe present disclosure, after the vitrified callus is preserved, the method preferably further includes that the preserved callus is recovered. The vitrified callus is preferably preserved for at least 24 h.
[25] In the present disclosure, a recovery method preferably includes the following steps: (a) the preserved callus is thawed in a water bath at 40°C;
[26] (b) the PVS2 solution in the thawed callus is removed by suction, and the thawed callus and an unloading solution are mixed for unloading to obtain an unload callus, where, the unloading solution includes a WPM basic culture medium and 410.4 g/L of sucrose, and the pH 1s 5.8; and
[27] (©) the unloading solution is removed by suction, and then the unloaded callus is transferred into an induction culture medium for recovery culture.
[28] In the present disclosure, before the unloading in step (b) is conducted, the method preferably further includes that disinfection is conducted on a surface of the cryogenic tube after the water bath, and then the cryogenic tube is placed on an ultra-clean workbench for the unloading, where, the disinfection is preferably conducted by wiping the surface with alcohol with a volume fraction of 75%. In the present disclosure, an added amount of the unloading solution is preferably the same as that of the loading solution. In the present disclosure, the unloading is preferably conducted at 25°C for 20 min. In the present disclosure, a preparation method of the unloading solution is not particularly limited. The method preferably includes that the components above are mixed, the pH is adjusted to 5.8, moist heat sterilization is conducted by using an autoclave at 121°C for 15 min, and then the unloading solution is placed at room temperature.
[29] The method for vitrification and ultra-low-temperature preservation of an embryogenic callus of Magnolia officinalis provided in the present disclosure is described in detail below in conjunction with embodiments, but it should not be understood that the protection scope of the present disclosure is limited to the embodiments.
[30] Example 1
[31] 1. Material selection: An embryogenic callus of Magnolia officinalis that was induced by zygotic embryos of mature seeds of Magnolia officinalis and cultured in a mixture including a WPM culture medium, 2 mg/L of 2,4-D, 0.25 mg/L of 6-BA, 1 g/L of polyvinylpyrrolidone (PVP), 1 g/L of a casein hydrolysate (CH), 40 g/L of sucrose and 3 g/L of phytagel under a pH of 5.8 in a dark environment at 25°C for 2 weeks was selected.
[32] 2. Measurement of 0.25 mL of the embryogenic callus of the Magnolia officinalis:
On an ultra-clean workbench, 1 mL of a WPM liquid culture medium was added into 1.5 mL of a sterilized centrifuge tube, and the lowest point of a concave liquid surface was flush with a 1 mL scale at this time. The embryogenic callus of the Magnolia officinalis in great condition was picked with tweezers and transferred into the 1.5 mL centrifuge tube until the lowest point of a concave liquid surface was flush with a 1.25 mL scale, where, the embryogenic callus of the Magnolia officinalis was measured in a volume of 0.25 mL.
[33] 3. Loading: A pipettor was equipped with a 1 mL cut-out blue pipette tip, 0.25 mL of the weighed embryogenic callus was transferred into a 2 mL cryogenic tube, the WPM liquid was sucked out, and 2 mL of a loading solution was added for loading for 20 min.
[34] 4. Vitrification: After the loading was completed, the loading solution was sucked out with the pipettor, and 2 mL of a PVS2 solution pre-cooled on ice was added for treatment on ice for 30 min.
[35] 5. Ultra-low-temperature preservation: After the vitrification was completed, the cryogenic tube was fixed to a cryogenic tube support. The cryogenic tube support was rapidly put into a liquid nitrogen tank for preservation for more than 24 h.
[36] 6. Thawing in a water bath at 40°C: The cryogenic tube support with the cryogenic tube preserved in liquid nitrogen for more than 24 h was taken out from the liquid nitrogen tank and rapidly inserted into a constant-temperature water bath at 40°C for thawing for 2 min.
[37] 7. Unloading: After the water bath, an outer surface of the cryogenic tube was wiped with 75% alcohol, the PVS2 solution was sucked out with the pipettor on the ultra-clean workbench, and then 2 mL of an unloading solution was added for treatment at 25°C for 20 min.
[38] 8. Washing and recovery culture: After the unloading was completed, the unloading solution was sucked out with the pipettor, and 2 mL of a WPM liquid culture medium was added for washing and then sucked out with the pipettor. After the washing was completed, 1 mL of a WPM liquid was added into the 2 mL cryogenic tube. The embryogenic callus was transferred into a 9 cm glass culture dish with 3 pieces of filter paper by using the pipettor equipped with the cut-out pipette tip. After a filter liquid was completely infiltrated, the embryogenic callus was transferred into a mixture including a WPM culture medium, 2 mg/L of 2,4-D, 0.25 mg/L of 6-BA, 1 g/L of PVP, 1 g/L of CH, 30 g/L of sucrose and 3 g/L of phytagel (phytagel) for recovery culture under a pH of 5.8 in a dark environment at 25°C.
[39] 190 tubes of the embryogenic callus after being frozen for two weeks were subjected to recovery culture 5 times. Results were shown in FIG. 1, where, 40 tubes of the embryogenic callus were separately recovered for the 1st, 2nd, 3rd and 4th times, 30 tubes of the embryogenic callus were recovered for the 5th time, and a survival rate after the recovery was 100%.
[40] 9. Identification of the survival rate and recovery ability and determination of the optimal time for treatment with the PVS2 solution
[41] A fluorescein diacetate (FDA) live cell fluorescent dye and a propidium iodide (PI) dead cell fluorescent dye were used for staining. Fluorescence changes of the embryogenic callus of the Magnolia officinalis at recovery time of 0 h, 1 day, 2 days, 3 days, 4 days, 5 days and 6 days were observed by using a laser confocal microscope. According to results, it was shown that the survival rate was first decreased during the recovery culture, a cell viability value was the lowest at 48 h and then was increased, and the viability value was close to that of a control at 7 days. It was concluded that the most accurate time point for determining the survival rate of the embryogenic callus of the Magnolia officinalis was 48 h.
[42] Then, the embryogenic callus was subjected to treatment with the PVS2 solution under a time gradient of 0 min, 5 min, 10 min, 30 min, 50 min, 70 min and 90 min, ultra- low-temperature preservation and recovery culture for 48 h, and the fluorescence and the fluorescence intensity were determined. Results showed that when the embryogenic callus was subjected to treatment with the PVS2 solution for 30 min and the recovery culture for 48 h, the fluorescence intensity was the highest, and that is, the activity was the highest.
Under observation of the ultra-low-temperature embryogenic callus for two weeks (as shown in FIG 2, where, a referred to a raw material subcultured for two weeks for ultra-low- temperature preservation; b referred to an embryogenic callus of Magnolia officinalis obtained after thawing and recovery culture for 48 h; c referred to an embryogenic callus of
Magnolia officinalis obtained after thawing and recovery culture for 2 weeks; and d referred to an embryogenic callus of Magnolia officinalis obtained after ultra-low-temperature regeneration and subculture for 2 weeks), it was concluded that the optimal time for vitrification of the embryogenic callus of the Magnolia officinalis was 30 min.
[43] 10. Subculture, differentiation and seedling formation of a regenerated embryogenic callus
[44] When the recovery culture was conducted for 1 month, a regenerated embryogenic callus on the filter paper was transferred into a mixture including a WPM culture medium, 2 mg/L of 2,4-D, 0.25 mg/L of 6-BA, 1 g/L of PVP, 1 g/L of CH, 30 g/L of sucrose and 3 g/L of phytagel (phytagel) for subculture without filter paper under a pH of 5.8 in a dark environment at 25°C. The culture medium was changed every two weeks. After the subculture was conducted twice, an embryogenic callus subcultured for two weeks was picked out and transferred into a WPM culture medium for differentiation. One month later, somatic embryos without the callus were produced, picked out and then put on a WPM culture medium. When roots and cotyledons grew, the somatic embryos were transferred into a tissue culture bottle for continuous culture. When the roots were developed, the somatic embryos were transferred into a seedling hole and finally prepared into a seedling bag. Recovery and seedling formation processes were shown in FIG. 3, where, a referred to a regenerated embryogenic callus of Magnolia officinalis after being preserved in liquid nitrogen; b referred to somatic embryos differentiated by the regenerated embryogenic callus; c referred to a somatic embryo; d referred to a tissue culture seedling of the Magnolia officinalis; e referred to a bottle of tissue culture seedlings of the Magnolia officinalis; and f referred to a Magnolia officinalis plant transplanted into a 1616 cm seedling bag.
The seedling rate was 100%, and the survival rate was 100%.
Claims (6)
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