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CN-121975623-A - Germplasm cryopreservation method suitable for bait microalgae

CN121975623ACN 121975623 ACN121975623 ACN 121975623ACN-121975623-A

Abstract

The invention relates to a germplasm freezing preservation method suitable for bait microalgae, belonging to the field of algae preservation, which comprises the steps of carrying out low-temperature domestication on microalgae in a logarithmic growth phase, and centrifugally collecting algae mud; mixing the algae mud with the protective solution, pretreating, mixing with the cryoprotectant with specific composition, cooling in gradient, preserving at-80deg.C, thawing, rapidly thawing in water bath, and gradient eluting the cryoprotectant with sucrose solution. The method successfully realizes the long-term stable preservation of a plurality of bait microalgae under the condition of-80 ℃ by optimizing the cryoprotectant combination and optimizing key parameters such as the protecting liquid, the balancing time, the thawing temperature and the like, has high survival rate after recovery, stable physiological property, low cost and convenient operation, and provides a new technical scheme for protecting and utilizing microalgae germplasm resources.

Inventors

  • ZHANG XIAOWEN
  • YE NAIHAO
  • WANG YITAO
  • ZHANG PENGYAN
  • FAN XIAO
  • XU DONG
  • SUN KE
  • Wang Lepu

Assignees

  • 中国水产科学研究院黄海水产研究所

Dates

Publication Date
20260505
Application Date
20260409

Claims (10)

  1. 1. A method for cryopreserving germplasm suitable for bait microalgae, the method comprising the steps of: S1, performing low-temperature domestication, namely placing microalgae in a logarithmic growth phase into a condition of 2-8 ℃ for dark treatment for 12-48 hours; S2, preprocessing, namely centrifuging the microalgae domesticated in the step S1, collecting algae mud, adding an equal volume of protection liquid into the algae mud, uniformly mixing, balancing for 10-30 minutes, and centrifuging to remove the protection liquid; S3, loading a cryoprotectant, namely adding the same volume of cryoprotectant into the algae mud pretreated in the step 2, balancing for 0-60 minutes in an ice bath at the temperature of 0 ℃, centrifuging to remove the cryoprotectant, adding the same volume of cryoprotectant again to resuspend the algae mud, and sub-packaging in a freezing tube; s4, gradient cooling and freezing, namely placing the freezing pipe in the step S3 in a refrigerator with the temperature of 2-8 ℃ for 20-40 minutes, then transferring the freezing pipe into the refrigerator with the temperature of-20 ℃ for 90-150 minutes, and finally transferring the freezing pipe into an ultralow temperature refrigerator with the temperature of-80 ℃ for preservation; And S5, thawing and recovering, namely taking out the cryopreservation tube which is preserved in the step S4, rapidly thawing in a water bath at 20-40 ℃ until the last ice crystal disappears, and transferring the thawed algae liquid into a culture medium for recovery culture after gradient elution of sucrose solution.
  2. 2. The method for cryopreserving germplasm of a bait microalgae according to claim 1, wherein the protective solution in the step S2 is a mixed solution of 2M glycerol and sucrose, and the concentration of the sucrose is 0.2M-0.6M.
  3. 3. The method for cryopreserving germplasm suitable for bait microalgae according to claim 2, wherein the cryoprotectant in the step S3 consists of dimethyl sulfoxide, ethylene glycol and proline, wherein the final concentration of the dimethyl sulfoxide is 5% by volume, the final concentration of the ethylene glycol is 10% by volume, and the final concentration of the proline is 10% by mass.
  4. 4. A method for cryopreservation of germplasm for bait microalgae according to claim 3, wherein the equilibration time in step S3 is 30 minutes.
  5. 5. A method for cryopreservation of germplasm for a bait microalgae according to claim 4, wherein the thawing temperature in step S5 is 20 ℃.
  6. 6. The germplasm cryopreservation method suitable for bait microalgae according to claim 5, wherein the protective solution in the step S2 is a mixed solution of 2M glycerol and 0.6M sucrose, the cryoprotectant in the step S3 consists of 5% dimethyl sulfoxide, 10% ethylene glycol and 10% proline in a volume ratio, the equilibrium time is 30 minutes, the thawing temperature in the step S5 is 20 ℃, and the method is suitable for cryopreservation of the Phillips qinghaosu.
  7. 7. The germplasm cryopreservation method suitable for bait microalgae according to claim 5, wherein the protective solution in the step S2 is a mixed solution of 2M glycerol and 0.2M sucrose, the balancing time in the step S3 is 0 minute, the thawing temperature in the step S5 is 40 ℃, and the method is suitable for cryopreservation of dinoflagellates such as dinoflagellates.
  8. 8. The germplasm cryopreservation method suitable for bait microalgae according to claim 5, wherein the protective solution in the step S2 is a mixed solution of 2M glycerol and 0.2M sucrose, the balancing time in the step S3 is 60 minutes, the thawing temperature in the step S5 is 20 ℃, and the method is suitable for cryopreservation of Phaeodactylum tricornutum.
  9. 9. The method for cryopreserving germplasm of a bait microalgae according to claim 5, wherein the gradient elution of the sucrose solution in the step S5 is specifically implemented by centrifuging the thawed microalgae solution, removing supernatant, sequentially adding 0.4M and 1.2M sucrose solution for treatment, centrifuging, and then rinsing with a culture medium.
  10. 10. A method for cryopreservation of germplasm for a bait micro-algae according to any one of claims 1-5, wherein the micro-algae is dinoflagellate, dinoflagellate or phaeodactylum tricornutum.

Description

Germplasm cryopreservation method suitable for bait microalgae Technical Field The application relates to the technical field of microbial preservation, in particular to a germplasm freezing preservation method suitable for bait microalgae. Background Microalgae are primary producers in aquatic ecosystems, many of which are also important economic cultivars, with high economic value. Germplasm resources are a source of innovation and development of the germplasm industry and become important strategic resources. The existing algae seed preservation mode is mainly a continuous passage preservation method, and the method has the problems of time consumption, labor consumption, easy genetic drift, physiological characteristic change and the like. Ultra-low temperature cryopreservation (e.g., liquid nitrogen, -196 ℃) is an ideal means of long-term preservation, but requires stable liquid nitrogen supply and special equipment, is costly, and limits its widespread use in ordinary laboratories. Therefore, development of a microalgae germplasm preservation method which is low in cost, convenient to operate and capable of achieving long-term stable preservation is needed, and particularly bait microalgae with important economic values for dinoflagellates such as dinoflagellates, globes and the like, phaeodactylum tricornutum and the like. Disclosure of Invention The application aims to provide a germplasm freezing preservation method suitable for bait microalgae, aiming at solving the problems of high microalgae preservation cost, easy occurrence of genetic drift and the like in the prior art. In order to achieve the above object, the present application provides the following technical solutions: a method of cryopreserving germplasm suitable for use in bait microalgae, the method comprising the steps of: S1, performing low-temperature domestication, namely placing microalgae in a logarithmic growth phase into a condition of 2-8 ℃ for dark treatment for 12-48 hours; S2, preprocessing, namely centrifuging the microalgae domesticated in the step S1, collecting algae mud, adding an equal volume of protection liquid into the algae mud, uniformly mixing, balancing for 10-30 minutes, and centrifuging to remove the protection liquid; S3, loading a cryoprotectant, namely adding the same volume of cryoprotectant into the algae mud pretreated in the step 2, balancing for 0-60 minutes in an ice bath at the temperature of 0 ℃, centrifuging to remove the cryoprotectant, adding the same volume of cryoprotectant again to resuspend the algae mud, and sub-packaging in a freezing tube; s4, gradient cooling and freezing, namely placing the freezing pipe in the step S3 in a refrigerator with the temperature of 2-8 ℃ for 20-40 minutes, then transferring the freezing pipe into the refrigerator with the temperature of-20 ℃ for 90-150 minutes, and finally transferring the freezing pipe into an ultralow temperature refrigerator with the temperature of-80 ℃ for preservation; And S5, thawing and recovering, namely taking out the cryopreservation tube which is preserved in the step S4, rapidly thawing in a water bath at 20-40 ℃ until the last ice crystal disappears, and transferring the thawed algae liquid into a culture medium for recovery culture after gradient elution of sucrose solution. Optionally, the protecting solution in the step S2 is a mixed solution of 2M glycerol and sucrose, and the concentration of the sucrose is 0.2M-0.6M. Alternatively, the cryoprotectant in step S3 consists of dimethyl sulfoxide, ethylene glycol and proline, wherein the final concentration of dimethyl sulfoxide is 5% (v/v), the final concentration of ethylene glycol is 10% (v/v), and the final concentration of proline is 10% (w/v). Alternatively, the equilibration time described in step S3 is 30 minutes. Alternatively, the thawing temperature described in step S5 is 20 ℃. Optionally, the protecting solution in the step S2 is a mixed solution of 2M glycerol and 0.6M sucrose, the cryoprotectant in the step S3 consists of 5% (v/v) dimethyl sulfoxide, 10% (v/v) ethylene glycol and 10% (w/v) proline, the balancing time is 30 minutes, the thawing temperature in the step S5 is 20 ℃, and the method is suitable for cryopreservation of the Phaerocarum qing. Optionally, the protection solution in the step S2 is a mixed solution of 2M glycerol and 0.2M sucrose, the balancing time in the step S3 is 0 minute, the thawing temperature in the step S5 is 40 ℃, and the method is suitable for cryopreservation of dinoflagellates such as dinoflagellates. Optionally, the protection solution in the step S2 is a mixed solution of 2M glycerol and 0.2M sucrose, the balancing time in the step S3 is 60 minutes, the thawing temperature in the step S5 is 20 ℃, and the method is suitable for the cryopreservation of Phaeodactylum tricornutum. Optionally, the gradient elution of the sucrose solution in the step S5 specifically comprises centrifuging the thawed algae solution, removing the sup