CN115918533A - Small droplet vitrification cryopreservation method for Guimu I grassiness - Google Patents
Small droplet vitrification cryopreservation method for Guimu I grassiness Download PDFInfo
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- CN115918533A CN115918533A CN202211509653.3A CN202211509653A CN115918533A CN 115918533 A CN115918533 A CN 115918533A CN 202211509653 A CN202211509653 A CN 202211509653A CN 115918533 A CN115918533 A CN 115918533A
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- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 17
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- 238000002791 soaking Methods 0.000 claims abstract description 13
- 244000177175 Typha elephantina Species 0.000 claims abstract description 12
- 244000025254 Cannabis sativa Species 0.000 claims abstract description 11
- 235000015696 Portulacaria afra Nutrition 0.000 claims abstract description 11
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- 238000012258 culturing Methods 0.000 claims abstract description 9
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- 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 21
- 239000007788 liquid Substances 0.000 claims description 21
- 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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- 238000011084 recovery Methods 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 235000011187 glycerol Nutrition 0.000 claims description 8
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- 239000002609 medium Substances 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
- 239000008272 agar Substances 0.000 claims description 4
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- 241001465754 Metazoa 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
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for cryopreservation of elephant grass in Guimu I by a droplet vitrification method, belonging to the technical field of plant genetic resource protection. The ultra-low temperature preservation method comprises the following steps: (1) Selecting axillary buds of the Guimu I grassiness, sterilizing and pre-culturing; (2) Cutting the pre-cultured axillary buds into sections, and soaking the axillary buds in a mixed solution of a vitrification solution containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4; (3) Transferring the soaked mixture into a vitrification solution drop containing hydrophobic nano calcium carbonate, quickly cooling and storing at ultralow temperature; and (4) recovering culture. The cryopreservation method of the elephantopus grass by the small droplet vitrification method has high survival rate and excellent germplasm preservation effect on the elephantopus grass.
Description
Technical Field
The invention relates to the technical field of plant genetic resource protection, in particular to a small droplet vitrification cryopreservation method for Guimu No. one elephant grass.
Background
The ultra-low 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 extremely low temperature below 80 ℃. Under the condition of ultralow temperature (generally liquid nitrogen and 196 ℃), almost all the metabolic activity and the growth process of the cells are stopped, the physiological activity is inhibited to the maximum extent, the occurrence of genetic variation is reduced, and therefore the stability of the biological material is maintained, but the cells still have the potential of vitality and morphogenesis. The ultra-low temperature preservation technology can avoid a plurality of unfavorable factors such as easy bacteria contamination and genetic variation in the traditional germplasm preservation process, and the germplasm preservation has the advantages of long-term property, stability, convenience and the like.
Depending on whether ice crystals are formed, cryopreservation is classified into two major categories, one is a conventional cryopreservation method, and the other is an cryopreservation method based on a vitrification method. The small-droplet vitrification method is a more optimized in-vitro preservation method based on the traditional vitrification method, namely, a high-efficiency ultralow-temperature preservation method which is used for treating a material by using vitrification liquid, then putting the material on aluminum foil paper containing vitrification liquid droplets and putting the aluminum foil paper into liquid nitrogen for freezing. But the problems of low survival rate, poor preservation effect and the like exist in the small-droplet vitrification cryopreservation method.
The Guimu I weevil grass belongs to high-yield gramineae forage grass, is obtained by carrying out sexual hybridization by taking dwarf weevil grass as a male parent and taking hybrid pennisetum alopecuroides as a female parent, belongs to novel hybrid forage grass, has the advantages of high yield, large leaf amount, stout stems, good quality, soft texture, good palatability, high utilization rate and the like, is very suitable for large-area planting, and is a good forage grass for herbivorous animals such as cattle, sheep, geese, ostrich, deer, fish and the like. In view of various excellent characteristics of the Guimu Xiangcao, the preservation of germplasm resources is necessary in the field for the sustainable development of agriculture.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preserving the Guimu No. I elephant grass by a small droplet vitrification method at an ultralow temperature.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preserving Guimu I elephant grass at ultralow temperature by a small droplet vitrification method comprises the following steps:
(1) Selecting axillary buds of the Guimu I grassiness, sterilizing and pre-culturing;
(2) Cutting the pre-cultured axillary buds into sections, and soaking the axillary buds in a mixed solution of a vitrification solution containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4;
(3) Transferring the soaked mixture into a vitrification solution drop containing hydrophobic nano calcium carbonate, quickly cooling and storing at ultralow temperature;
(4) And (5) recovering culture.
Preferably, the disinfection in step (1) is soaking with 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 pre-culture medium in the step (1) is MS solid medium containing 0.4mol/L sucrose and 2mol/L glycerol, and the culture time is 4d.
Preferably, the vitrification liquid is a 0.4M sucrose solution containing 30% by volume of glycerol, 15% of ethylene glycol and 15% of DMSO; 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 to 0.6g/L.
More preferably, the preparation step 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.
More preferably, the nano calcium carbonate has a particle size of not more than 50nm; the adding amount of the stearic acid is 2 percent of the mass of the nano calcium carbonate.
Preferably, the temperature of the soaking treatment in the step (2) is 0 ℃ and the time is 50min.
Preferably, the specific steps of recovering the culture in step (4) include: and (3) thawing the axillary buds of the Guimu I Xiangcao preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu I Xiangcao plant.
More preferably, the thawing is to keep the temperature of 35-40 ℃ for 2-4 min; the unloaded liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery culture medium is an MS culture medium containing 0.1mg/L of 6-BA,0.05mg/LIAA,30g/L of sucrose and 7.0g/L of agar.
More preferably, after inoculation into the recovery medium, the culture is performed for 1d in dark and then for 16h/d in light.
The invention has the following beneficial technical effects:
the invention provides a method for ultralow temperature preservation of Guimu No. I grassiness by a droplet vitrification method, which comprises the steps of selecting axillary buds of Guimu No. I grassiness as preservation objects, pre-culturing to fully stimulate the activity of the axillary bud cells of Guimu No. I grassiness, and adding hydrophobic nano calcium carbonate into vitrification liquid in an ultralow temperature preservation mode of the droplet vitrification method to play a better protection role on the axillary bud cells of Guimu No. I grassiness, so that the survival rate of the axillary buds of Guimu No. I grassiness after thawing is obviously improved.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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.
Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 open-ended terms that mean including, but not limited to.
The invention provides a small droplet vitrification cryopreservation method for Guimu No. I grassiness, which comprises the following steps:
(1) Selecting axillary buds of the Guimu I grassiness, sterilizing and pre-culturing;
(2) Cutting the pre-cultured axillary buds into sections, and soaking the axillary buds in a mixed solution of a vitrification solution containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4;
(3) Transferring the soaked mixture into a vitrification solution drop containing hydrophobic nano calcium carbonate, quickly cooling and storing at ultralow temperature;
(4) And (5) recovering the culture.
According to the method, the axillary bud tips of the Guimu I elephantopus grass are selected as the ultralow-temperature preservation objects, and compared with the traditional scheme of preserving the stem tips by using a droplet vitrification method, the axillary bud tips have higher differentiation rate and are easier to obtain plants.
In an ultralow temperature environment, cytoplasm of the Guimu I elephant grass axillary bud cell can form a vitrification state under the protection of vitrification liquid containing hydrophobic nano calcium carbonate, free water content in the cytoplasm is low due to early-stage soaking treatment, and after the hydrophobic nano calcium carbonate is added into the vitrification liquid, micro ice crystals are not easy to form in the ultralow temperature environment, destructive power to the cell is low, and the cell can survive at a low temperature.
Preferably, the disinfection in step (1) is soaking with 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 not only ensure to eliminate the pollution problem of the axillary buds of the Guimu I grassiness, but also can not cause damage to the axillary buds of the Guimu I grassiness.
Preferably, the pre-culture medium in the step (1) is MS solid medium containing 0.4mol/L sucrose and 2mol/L glycerol, and the culture time is 4d.
The invention strictly controls the pre-culture time, thereby not only ensuring that the axillary buds of the Guimu No. I elephant grass stored at ultralow temperature have sufficient activity, but also avoiding the over-mature cells caused by the overlong pre-culture time, wherein the over-mature cells contain large vacuoles and have higher free water content, and dendritic ice crystals which damage the cell membrane structure are more easily formed in the ultralow temperature environment to cause cell death.
Preferably, the vitrification solution is a 0.4M sucrose solution containing 30% by volume of glycerol, 15% of ethylene glycol and 15% of DMSO; 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 to 0.6g/L.
More preferably, the preparation step 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 stearic acid is selected for modification during hydrophobic modification, firstly, the stearic acid modified nano calcium carbonate is better dispersed in the vitrification solution, and secondly, the stearic acid modified nano calcium carbonate is easier to enter axillary bud cells of the Guimu I grassiness, so that the Guimu I grassiness axillary bud cells are protected under the ultralow temperature condition, and toxicity can not be generated on the Guimu I grassiness axillary buds after thawing.
More preferably, the nano calcium carbonate has a particle size of not more than 50nm; the adding amount of the stearic acid is 2 percent of the mass of the nano calcium carbonate.
Preferably, the temperature of the soaking treatment in the step (2) is 0 ℃ and the time is 50min.
Preferably, the specific steps of recovering the culture in step (4) include: and (3) thawing the axillary buds of the Guimu I grassiness which is preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu I grassiness plant.
More preferably, the thawing is to keep the temperature of 35-40 ℃ for 2-4 min; the unloaded liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery culture medium is an MS culture medium containing 0.1mg/L of 6-BA,0.05mg/LIAA,30g/L of sucrose and 7.0g/L of agar.
The temperature condition selected during thawing can avoid the phenomenon of re-freezing in cells caused by slow temperature rise in the thawing process, and can keep the cell activity of the axillary buds of the Guimu I grassiness to the maximum extent.
More preferably, after inoculation into the recovery medium, the culture is performed for 1d in the dark and then for 16h/d in the light.
The vitrification liquids used in the examples of the present invention and the comparative examples were 0.4M sucrose solutions containing 30% by volume of glycerin, 15% of ethylene glycol and 15% of DMSO.
Example 1
(1) Preparing stearic acid modified nano calcium carbonate: placing nano calcium carbonate with the particle size not more than 50nm in a beaker, adding water, stirring and dispersing, then placing in 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, and grinding until the particle size is not more than 100nm to obtain stearic acid modified nano calcium carbonate;
(2) Pre-culturing: selecting axillary buds of the Guimu I grassiness, washing with sterile water, adding the axillary buds into 0.1g/100mL mercuric chloride solution for soaking for 25min, then adding the axillary buds into 2g/100mL sodium hypochlorite solution for soaking for 20min, washing with the sterile water, sucking water, transferring the axillary buds into an MS solid culture medium containing 0.4mol/L sucrose and 2mol/L glycerol for pre-culture under the culture conditions of 2000Lux illumination for 14h/d, the temperature of 25 +/-2 ℃, the humidity of 75 +/-5% and the culture for 4d;
(3) And (3) treating the glass melting liquid: cutting axillary buds of pre-cultured elephantopus grass I into 0.4-0.5 mm axillary bud segments, adding the axillary buds into a mixed solution of 0.5g/L stearic acid modified nano calcium carbonate obtained in the step (1) and sterile water in a volume ratio of 4;
(4) And (4) ultra-low temperature preservation: transferring the axillary bud segments soaked in the step (3) to an aluminum foil strip dropped with a vitrification liquid drop containing 0.5g/L of stearic acid modified nano calcium carbonate obtained in the step (1), entering liquid nitrogen for 2s, and then transferring to a freezing tube filled with liquid nitrogen for ultralow-temperature preservation;
(5) And (3) recovery culture: after being preserved at ultralow temperature for 1 day, 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 of sucrose for unloading for 20min, finally transferred into an MS culture medium containing 0.1mg/L of 6-BA,0.05mg/LIAA,30g/L of sucrose and 7.0g/L of agar, and subjected to dark culture for 1 day under the conditions that the temperature is 25 +/-2 ℃ and the humidity is 75 +/-5 percent, and then subjected to 2000Lux illumination for 16 hours/day for culture, so as to obtain the elephant grass plant of Guimu I. During recovery culture, 100 segments of ultralow-temperature preserved axillary buds of the Guimu I grassiness are selected, the number of growing plants of the Guimu I grassiness is recorded, and the survival rate (%) is calculated and is not equal to the number of the plants of the Guimu I grassiness/100 multiplied by 100%.
Comparative example 1
Compared with the embodiment 1, the difference is only 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 is replaced by the vitrification liquid containing 0.5g/L nano calcium carbonate (the particle size is not more than 50 nm) in the subsequent steps (3) and (4).
Comparative example 2
Compared with the embodiment 1, the difference is only that the addition of the stearic acid modified nano calcium carbonate is omitted.
Comparative example 3
The only difference compared to example 1 is that the preculture time was 2d.
Comparative example 4
The only difference compared to example 1 is that the preculture time was 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 segments of the first herb of Guimu No. 1 preserved at ultralow temperature in each group of the example 1 and the comparative examples 1 to 4 is counted, and the statistical result is shown in the table 1.
TABLE 1 survival rate of ultra-low temperature preserved Guimu I grassiness axillary bud
| Recovery of culture number | Number of survivals | Survival rate | |
| 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％ |
From the survival rate recorded in table 1, compared with the ultralow-temperature storage condition without adding nano calcium carbonate or adding unmodified nano calcium carbonate, the survival rate of the underarm buds of the cinnamomum gracile habenne stored at ultralow temperature is remarkably improved after hydrophobic modified nano calcium carbonate is added; meanwhile, the pre-culture time also has great influence on the survival rate of axillary buds of the Guimu I grassiness; in addition, the reasonable selection of the thawing temperature can also play a positive role in the survival rate of the axillary buds of the Guimu I grassiness.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (8)
1. A method for preserving Guimu No. I grassiness at ultralow temperature by a droplet vitrification method is characterized by comprising the following steps:
(1) Selecting axillary buds of the Guimu I elephantopus grass, sterilizing and pre-culturing;
(2) Cutting the pre-cultured axillary buds into sections, and soaking the axillary buds in a mixed solution of a vitrification solution containing hydrophobic nano calcium carbonate and water according to a volume ratio of 4;
(3) Transferring the soaked mixture into a vitrification solution drop containing hydrophobic nano calcium carbonate, quickly cooling and storing at ultralow temperature;
(4) And (5) recovering the culture.
2. The method for performing small-droplet vitrification cryopreservation on the Guimu No. I elephant grass according to claim 1, wherein the disinfection in the step (1) is a soaking in mercuric chloride solution and sodium hypochlorite solution in sequence.
3. The method for performing droplet vitrification cryopreservation on elephant grass herb No. 1, which is characterized in that the pre-culture medium in the step (1) is MS solid medium containing 0.4mol/L sucrose and 2mol/L glycerin, and the culture time is 4 days.
4. The method for small droplet vitrification cryopreservation of elephant grass, no. 1, wherein the vitrification solution is a 0.4M sucrose solution containing 30% by volume of glycerin, 15% of ethylene glycol and 15% of dmso; the hydrophobic nano calcium carbonate is stearic acid modified nano calcium carbonate; the addition amount of the hydrophobic nano calcium carbonate in the glass liquid containing the hydrophobic nano calcium carbonate is 0.5-0.6 g/L.
5. The method for performing small-droplet vitrification cryopreservation on the Guimu No. I elephant grass according to claim 4, wherein the preparation step 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.
6. The method for performing small droplet vitrification cryopreservation on the Guimu No. I elephant grass according to claim 1, wherein the temperature of the soaking treatment in the step (2) is 0 ℃ and the time is 50min.
7. The method for performing small droplet vitrification cryopreservation on the Guimu No. I grassiness according to claim 1, wherein the specific steps for restoring the culture in the step (4) comprise: and (3) thawing the axillary buds of the Guimu I grassiness which is preserved at ultralow temperature, unloading, then inoculating into a recovery culture medium, and culturing to obtain the Guimu I grassiness plant.
8. The small droplet vitrification cryopreservation method of the sweet osmanthus grass I according to claim 7, wherein the thawing is performed at 35-40 ℃ for 2-4 min; the unloaded liquid is MS liquid culture medium containing 1.2mol/L sucrose; the recovery medium is MS medium containing 0.1mg/L6-BA,0.05mg/L IAA,30g/L sucrose and 7.0g/L agar.
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