CN115918533B - Ultralow-temperature preservation method for Ipomoea batatas droplet vitrification method - Google Patents
Ultralow-temperature preservation method for Ipomoea batatas droplet vitrification method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000004321 preservation Methods 0.000 title claims abstract description 31
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- 235000002678 Ipomoea batatas Nutrition 0.000 title description 10
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 102
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 51
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- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical group CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 25
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 25
- 239000008117 stearic acid Substances 0.000 claims description 25
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 23
- 229930006000 Sucrose Natural products 0.000 claims description 16
- 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 16
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- 238000010257 thawing Methods 0.000 claims description 13
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- 238000011084 recovery Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 229960002523 mercuric chloride Drugs 0.000 claims description 6
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 4
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- 238000000338 in vitro Methods 0.000 description 2
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- 241000251468 Actinopterygii Species 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000008734 Bergera koenigii Nutrition 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 241000037488 Coccoloba pubescens Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000002393 Murraya koenigii Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
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- 241000272534 Struthio camelus Species 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Abstract
The invention discloses a method for ultralow-temperature preservation of Guimu-Yi-Cao by a droplet vitrification method, and belongs to the technical field of plant genetic resource protection. The ultralow temperature preservation method comprises the following steps: (1) Axillary buds of Ixeris sonchifolia are selected, sterilized and pre-cultured; (2) Cutting the axillary buds after preculture, and soaking the axillary buds with a mixed solution of vitrification liquid containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4:1; (3) Transferring the soaked solution into vitrification liquid drops containing hydrophobic nano calcium carbonate, rapidly cooling, and preserving at ultralow temperature; and (4) recovering culture. The ultralow-temperature preservation method of the Guimu grass by the small-drop vitrification method has high survival rate and excellent germplasm preservation effect on Guimu grass.
Description
Technical Field
The invention relates to the technical field of plant genetic resource protection, in particular to a method for ultralow-temperature preservation of Mesona chinensis by a small-drop vitrification method.
Background
Ultralow temperature preservation is a product combining in-vitro preservation and low temperature biology, and refers to a whole set of biological technology for preserving germplasm resources at an ultralow temperature below-80 ℃. Under ultralow temperature (liquid nitrogen, -196 deg.c) almost all the cell metabolism and growth process are stopped, so that physiological activity is inhibited to the maximum extent and genetic variation is reduced, and thus the stability of biological material is maintained, but the cell still has activity and morphogenetic potential. The ultralow temperature preservation technology can avoid a plurality of adverse factors such as easy bacteria contamination and easy genetic variation in the traditional germplasm preservation process, and the germplasm preservation has the advantages of long-term property, stability, convenience and the like.
According to whether ice crystals are formed, the ultra-low temperature preservation is classified into two main types, one is a conventional ultra-low temperature preservation method, and the other is an ultra-low temperature preservation method based on a vitrification method. The droplet vitrification method is a more optimized in-vitro preservation method based on the traditional vitrification method, namely, a vitrification liquid is used for processing materials, and then the materials are placed on aluminum foil paper containing vitrification droplets and put into liquid nitrogen for freezing and preserving. However, the ultralow temperature preservation by the droplet vitrification method has the problems of low survival rate, poor preservation effect and the like.
The grazing first-grade grassiness belongs to high-yield grazing grassiness, is obtained by sexual hybridization by taking the pennisetum as a male parent and taking the hybrid pennisetum as a female parent, belongs to novel hybrid grassiness, has the advantages of high yield, large leaf quantity, thick stalk, good quality, soft texture, good palatability, high utilization rate and the like, is very suitable for large-area planting, and is good forage grass for herbivorous animals such as cattle, sheep, geese, ostrich, deer, fish and the like. In view of the various excellent properties of the grassiness of Gui Murraya koenigii, it is necessary in the art to preserve its germplasm resources for sustainable development of agriculture.
Disclosure of Invention
In order to solve the problems, the invention provides a method for ultralow-temperature preservation of the Meimao Yi-Cao droplet by a vitrification method.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for ultralow temperature preservation of a Guimu No. I grassiness droplet vitrification method comprises the following steps:
(1) Axillary buds of Ixeris sonchifolia are selected, sterilized and pre-cultured;
(2) Cutting the axillary buds after preculture, and soaking the axillary buds with a mixed solution of vitrification liquid containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4:1;
(3) Transferring the soaked solution into vitrification liquid drops containing hydrophobic nano calcium carbonate, rapidly cooling, and preserving at ultralow temperature;
(4) And (5) recovering culture.
Preferably, the disinfection in the step (1) is soaking by using a mercuric chloride solution and a sodium hypochlorite solution in sequence.
More preferably, the concentration of the mercuric chloride solution is 0.1g/100mL; the concentration of the sodium hypochlorite solution is 2g/100mL.
Preferably, the preculture medium in step (1) is an MS solid medium containing 0.4mol/L sucrose and 2mol/L glycerol for a period of 4d.
Preferably, the vitrification solution is a 0.4M sucrose solution containing 30% glycerol, 15% ethylene glycol, and 15% dmso by volume; the hydrophobic nano calcium carbonate is stearic acid modified nano calcium carbonate; the addition amount of the hydrophobic nano calcium carbonate in the vitrification liquid containing the hydrophobic nano calcium carbonate is 0.5-0.6 g/L.
More preferably, the preparation steps of the stearic acid modified nano calcium carbonate include: dispersing nano calcium carbonate in water, adding stearic acid, ultrasonic treatment at 60-80 deg.c, filtering, drying and crushing to obtain stearic acid modified nano calcium carbonate.
More preferably, the particle size of the nano calcium carbonate is not more than 50nm; the addition amount of the stearic acid is 2% of the mass of the nano calcium carbonate.
Preferably, the soaking treatment in the step (2) is performed at a temperature of 0 ℃ for 50min.
Preferably, the specific step of recovering the culture in step (4) comprises: thawing the axillary buds of the Guimu first grass preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu first grass plant.
More preferably, the thawing is kept at a temperature of 35-40 ℃ for 2-4 min; the unloading liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery medium was MS medium containing 0.1mg/L6-BA,0.05mg/LIAA,30g/L sucrose and 7.0g/L agar.
More preferably, after inoculation into the recovery medium, the culture is performed in the dark for 1d and then in the light for 16 h/d.
The beneficial technical effects of the invention are as follows:
the invention provides a method for ultralow temperature preservation of a small-drop vitrification method of Guimu No. I grassroots, which comprises the steps of selecting axillary buds of Guimu No. I grassroots as preservation objects, pre-culturing, fully exciting the activity of the axillary buds of Guimu No. I grassroots, adding hydrophobic nano calcium carbonate into vitrification liquid in an ultralow temperature preservation mode of the small-drop vitrification method, and playing a better role in protecting the axillary buds of Guimu No. I grassroots, so that the survival rate of the axillary buds of Guimu No. I grassroots after thawing is obviously improved.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention provides a method for ultralow-temperature preservation of a Guimu grass droplet by a vitrification method, which comprises the following steps:
(1) Axillary buds of Ixeris sonchifolia are selected, sterilized and pre-cultured;
(2) Cutting the axillary buds after preculture, and soaking the axillary buds with a mixed solution of vitrification liquid containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4:1;
(3) Transferring the soaked solution into vitrification liquid drops containing hydrophobic nano calcium carbonate, rapidly cooling, and preserving at ultralow temperature;
(4) And (5) recovering culture.
The invention selects the axillary bud tip of the Ixeris sonchifolia as an ultralow temperature preservation object, and compared with the traditional scheme of preserving the stem tip by using a droplet vitrification method, the axillary bud tip has higher differentiation rate and is easier to obtain plants.
In the ultralow temperature environment, the cytoplasm of the Ixeris sonchifolia axillary bud cell can form a vitrified state under the protection of the vitrified liquid containing the hydrophobic nano calcium carbonate, and the free water content in the cytoplasm is low due to the early soaking treatment, and after the hydrophobic nano calcium carbonate is added into the vitrified liquid, tiny ice crystals are not easy to form in the ultralow temperature environment, the damage to the cell is smaller, and the Ixeris sonchifolia axillary bud cell can survive under the low temperature.
Preferably, the disinfection in the step (1) is soaking by using a mercuric chloride solution and a sodium hypochlorite solution in sequence.
More preferably, the concentration of the mercuric chloride solution is 0.1g/100mL; the concentration of the sodium hypochlorite solution is 2g/100mL.
The disinfection condition adopted by the invention can ensure that the pollution problem of the axillary buds of the Guimu grass can be eliminated, and the axillary buds of the Guimu grass can not be damaged.
Preferably, the preculture medium in step (1) is an MS solid medium containing 0.4mol/L sucrose and 2mol/L glycerol for a period of 4d.
By strictly controlling the pre-culture time, the invention not only ensures that the axillary buds of the Ipomoea batatas preserved at ultralow temperature have sufficient activity, but also avoids the cell death caused by overmaturation of cells due to overlong pre-culture time, the overmature cells contain large vacuoles, the free water content is higher, and tree-shaped ice crystals which destroy cell membrane structures are easier to form in an ultralow temperature environment.
Preferably, the vitrification solution is a 0.4M sucrose solution containing 30% glycerol, 15% ethylene glycol, and 15% dmso by volume; the hydrophobic nano calcium carbonate is stearic acid modified nano calcium carbonate; the addition amount of the hydrophobic nano calcium carbonate in the vitrification liquid containing the hydrophobic nano calcium carbonate is 0.5-0.6 g/L.
More preferably, the preparation steps of the stearic acid modified nano calcium carbonate include: dispersing nano calcium carbonate in water, adding stearic acid, ultrasonic treatment at 60-80 deg.c, filtering, drying and crushing to obtain stearic acid modified nano calcium carbonate.
According to the invention, stearic acid is selected for modification during hydrophobic modification, firstly, the stearic acid modified nano calcium carbonate is better dispersed in vitrification liquid, and secondly, the stearic acid modified nano calcium carbonate is easier to enter into the armpit cells of the Ipomoea batatas, so that the armpit cells of the Ipomoea batatas are protected under the ultralow temperature condition, and toxicity to the armpit cells of the Ipomoea batatas is avoided after thawing.
More preferably, the particle size of the nano calcium carbonate is not more than 50nm; the addition amount of the stearic acid is 2% of the mass of the nano calcium carbonate.
Preferably, the soaking treatment in the step (2) is performed at a temperature of 0 ℃ for 50min.
Preferably, the specific step of recovering the culture in step (4) comprises: thawing the axillary buds of the Guimu first grass preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu first grass plant.
More preferably, the thawing is kept at a temperature of 35-40 ℃ for 2-4 min; the unloading liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery medium was MS medium containing 0.1mg/L6-BA,0.05mg/LIAA,30g/L sucrose and 7.0g/L agar.
The temperature condition selected in the thawing process can avoid the phenomenon of re-icing in cells caused by slow temperature rise in the thawing process, and can keep the cell activity of the axillary buds of the Gui Mu Yi Cao to the greatest extent.
More preferably, after inoculation into the recovery medium, the culture is performed in the dark for 1d and then in the light for 16 h/d.
The vitrification solution used in the examples and comparative examples of the present invention was a 0.4M sucrose solution containing 30% glycerol, 15% ethylene glycol and 15% dmso by volume.
Example 1
(1) Preparing stearic acid modified nano calcium carbonate: placing nano calcium carbonate with the particle size not exceeding 50nm into a beaker, adding water, stirring and dispersing, placing into a constant-temperature ultrasonic cleaning machine, firstly keeping the temperature to 70 ℃, adding stearic acid with the mass fraction of 2% of the nano calcium carbonate, carrying out ultrasonic treatment for 20min, then filtering, drying, grinding to the particle size not exceeding 100nm, and obtaining stearic acid modified nano calcium carbonate;
(2) Pre-culturing: the axillary buds of Ipomoea batatas are selected, washed by sterile water, then added into 0.1g/100mL of mercuric chloride solution for soaking for 25min, then added into 2g/100mL of sodium hypochlorite solution for soaking for 20min, washed by sterile water, absorbed by water, transferred into an MS solid culture medium containing 0.4mol/L sucrose and 2mol/L glycerin for preculture, and cultured for 4d under the conditions of 2000Lux illumination for 14h/d at the temperature of 25+/-2 ℃ and the humidity of 75+/-5%;
(3) And (3) vitrification liquid treatment: cutting axillary buds of Ipomoea batatas after preculture into axillary bud sections with the size of 0.4-0.5 mm, adding the axillary buds into a mixed solution which is prepared by mixing 0 ℃ vitrification solution containing stearic acid modified nano calcium carbonate obtained in the step (1) and sterile water according to the volume ratio of 4:1, and soaking for 50min;
(4) And (5) ultralow temperature preservation: transferring the axillary bud segment soaked in the step (3) into an aluminum foil strip dripped with glass liquid drops containing 0.5g/L stearic acid modified nano calcium carbonate obtained in the step (1), entering liquid nitrogen for 2s, and then transferring into a freezing tube filled with liquid nitrogen for ultralow temperature preservation;
(5) Recovery culture: after being preserved for 1d at ultralow temperature, the aluminum foil strips are taken out, put into sterile water at 40 ℃ for thawing for 3min, then transferred into an MS liquid culture medium containing 1.2mol/L sucrose for unloading for 20min, finally transferred into an MS culture medium containing 0.1mg/L6-BA,0.05mg/LIAA,30g/L sucrose and 7.0g/L agar, and subjected to dark culture for 1d at the temperature of 25+/-2 ℃ and the humidity of 75+/-5%, and then subjected to 2000Lux illumination for 16h/d culture, so that the Ipomoea batatas Var. And selecting 100 ultralow-temperature preserved axillary bud segments of the Guimu first grass, recording the number of the plant growing into the Guimu first grass, calculating the survival rate (%), wherein the survival rate (%) =the number of the plant growing into the Guimu first grass/100×100%.
Comparative example 1
The difference compared with example 1 is that the step of stearic acid modified nano calcium carbonate is omitted, and the vitrification liquid containing 0.5g/L stearic acid modified nano calcium carbonate in the subsequent steps (3) and (4) is replaced with a vitrification liquid containing 0.5g/L nano calcium carbonate (particle size not more than 50 nm).
Comparative example 2
The only difference compared to example 1 is that the addition of stearic acid modified nano calcium carbonate was omitted.
Comparative example 3
The only difference compared to example 1 is that the preculture time is 2d.
Comparative example 4
The only difference compared to example 1 is that the preculture time is 5d.
Comparative example 5
The only difference compared to example 1 is that the thawing temperature is 25 ℃.
The survival rate of the axillary bud segment of the Guimu grass of each group of the example 1 and the comparative examples 1 to 4 is counted, and the counted results are shown in the table 1.
Table 1 survival rates of groups of ultra-Low temperature preserved axillary buds of Guimu No. I grasses
| Recovery of culture number | Number of survivors | Survival rate of | |
| Example 1 | 100 | 93 | 93％ |
| Comparative example 1 | 100 | 89 | 89％ |
| Comparative example 2 | 100 | 83 | 83％ |
| Comparative example 3 | 100 | 84 | 84％ |
| Comparative example 4 | 100 | 85 | 85％ |
| Comparative example 5 | 100 | 87 | 87％ |
As can be seen from the survival rate recorded in table 1, compared with the ultralow temperature preservation condition without adding nano calcium carbonate or without adding unmodified nano calcium carbonate, the survival rate of the axillary buds of the Gui grass preserved at ultralow temperature is remarkably improved after the hydrophobic modified nano calcium carbonate is added; meanwhile, the pre-culture time has a great influence on the survival rate of the axillary buds of the Guimu grass; in addition, the reasonable selection of the thawing temperature can also have positive effects on the survival rate of the axillary buds of the Ipomoea batatas.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (3)
1. The method for ultralow-temperature preservation of the Meimao-first-grade grassiness droplet vitrification method is characterized by comprising the following steps of:
(1) Axillary buds of Ixeris sonchifolia are selected, sterilized and pre-cultured;
(2) Cutting the axillary buds after preculture, and soaking the axillary buds with a mixed solution of vitrification liquid containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4:1;
(3) Transferring the soaked solution into vitrification liquid drops containing hydrophobic nano calcium carbonate, rapidly cooling, and preserving at ultralow temperature;
(4) Recovering and culturing;
the culture medium for the preculture in the step (1) is an MS solid culture medium containing 0.4mol/L sucrose and 2mol/L glycerol, and the culture time is 4d;
the vitrification solution is a 0.4M sucrose solution containing 30 percent of glycerol, 15 percent of ethylene glycol and 15 percent of DMSO by volume fraction; the hydrophobic nano calcium carbonate is stearic acid modified nano calcium carbonate; the addition amount of the hydrophobic nano calcium carbonate in the vitrification liquid containing the hydrophobic nano calcium carbonate is 0.5-0.6 g/L;
the preparation method of the stearic acid modified nano calcium carbonate comprises the following steps: dispersing nano calcium carbonate in water, adding stearic acid, performing ultrasonic treatment at 60-80 ℃, filtering, drying and crushing to obtain stearic acid modified nano calcium carbonate;
the specific steps of the recovery culture in the step (4) comprise: thawing the axillary buds of the Guimu first grass preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu first grass plant;
the thawing is carried out for 2-4 min at the temperature of 35-40 ℃; the unloading liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery medium was MS medium containing 0.1mg/L6-BA,0.05mg/L IAA,30g/L sucrose and 7.0g/L agar.
2. The method for ultralow temperature preservation of cassia nomamea small-drop grasses by vitrification according to claim 1, wherein the disinfection in the step (1) is soaking by using a mercuric chloride solution and a sodium hypochlorite solution in sequence.
3. The method for ultralow temperature preservation of the small-drop vitrification method of cassia nomamea, according to claim 1, wherein the soaking treatment in the step (2) is carried out at a temperature of 0 ℃ for 50min.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211509653.3A CN115918533B (en) | 2022-11-29 | 2022-11-29 | Ultralow-temperature preservation method for Ipomoea batatas droplet vitrification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211509653.3A CN115918533B (en) | 2022-11-29 | 2022-11-29 | Ultralow-temperature preservation method for Ipomoea batatas droplet vitrification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115918533A CN115918533A (en) | 2023-04-07 |
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| CN106460022B (en) * | 2014-06-24 | 2018-06-05 | 加州大学董事会 | The production method of xyloside xylitol oligomer |
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