Method for vitrification ultra-low temperature preservation of sporangium of endangered pteridium huperzia serrata

Abstract

The invention relates to the technical field of germplasm preservation, and particularly provides an ultralow-temperature preservation method of huperzia serrata. Comprising the following steps: by taking huperzia serrata sporangium as a preservation material, loading the sporangium dried for 30 minutes into a cryopreservation tube, treating the cryopreservation tube in an LS loading liquid at the room temperature of 25 DEG C for 25 minutes, and removing the LS loading liquid; transferring into vitrification protection liquid PVS for ice bath treatment for 30 minutes, replacing with pre-cooled 4 DEG C fresh PVS2 protection liquid, and quickly putting into liquid nitrogen for preservation. After preservation is finished, the cryopreservation tube is cryopreserved in liquid hydrogen for 24 h and then taken out, the cryopreservation tube is immediately put into a water bath at the temperature of 37 DEG C to be quickly unfrozen for 2 min, the cryopreservation tube is placed on a super clean bench to be soaked in US unloading liquid three times and 5 min per time, and finally the cryopreservation tube is washed three times with sterilized purified water. The effective protective agent is applied to the ultralow-temperature preservation method, the survival rate of huperzia serrata after ultralow-temperature preservation is improved, the diversity of pteridium aquilinum germplasm ultralow-temperature preservation materials is enriched, and a new thought is provided for searching for pteridium aquilinum germplasm ultralow-temperature preservation.

Description

Technical Field

[0001] The present invention belongs to the technical field of germplasm preservation, and relates to a vitrification cryopreservation method for Huperzia serrata Background Art

[0002] Huperzia serrata (Thunb.) Trevis., scientifically named Huperzia serrata, is a perennial rare and endangered medicinal herbaceous fern of the genus Huperzia in the family Huperziaceae. Huperzia serrata has been used as a herbal medicine for treating traumatic injuries, stasis swelling and pain, schizophrenia and other diseases in Chinese folk. Especially, the secondary metabolite huperzine A contained in it has attracted much attention from the global medical community for its wide application in the treatment of senile memory decline, cognitive impairment, and Alzheimer's disease. At present, scholars at home and abroad have not obtained a substitute with better activity than natural huperzine A for the synthesis of huperzine A, derivatives and analogues. Huperzia serrata plants are short, grow extremely slowly, have small biomass, and have limited wild resources. And the uncontrolled harvesting of the natural resources of Huperzia serrata by humans has caused the exhaustion and endangerment of resources. Therefore, effectively preserving its germplasm resources is crucial for maintaining species diversity, maintaining ecological balance, and future resource needs

[0003] Plant cryopreservation is a new biotechnology applied to plant germplasm preservation, which is one of the research hotspots in cryobiology and the only method that can safely preserve plant germplasm resources for a long time at present

[0004] Therefore, it is very important to optimize the cryopreservation system of Huperzia serrata sporangia to enhance its vitality and maintain its viability. How to apply effective cryoprotectants to the main steps such as cryo-loading treatment, dehydration treatment, and rapid thawing in cryopreservation, and effectively optimize the cryopreservation system are the key points for the success of the cryopreservation technology of Huperzia serrata Summary of the Invention

[0005] The purpose of the present invention is to provide a vitrification cryopreservation method for Huperzia serrata. This method can make up for the deficiencies of the existing Huperzia serrata germplasm resource preservation technology, improve the survival rate of Huperzia serrata after cryopreservation, enrich the diversity of cryopreservation materials for fern germplasm, and provide new ideas for seeking cryopreservation of fern plant germplasm

[0006] The purpose of the present invention is achieved through the following technical solutions

[0007] A vitrification cryopreservation method for Huperzia serrata uses Huperzia serrata sporangia as preservation materials, and the specific steps are as follows

[0008] (1) Material acquisition: Select plump, uniform, and healthy sporangia, and use the bromothymol blue method to measure the initial viability of the sporangia. Select sporangia with an initial viability ≥ 90% as preservation materials

[0009] (2) Screening for appropriate water content treatment: Place the sporangia selected in step (1) into a 2 mL centrifuge tube. Open the lid of the centrifuge tube and place the centrifuge tube in a desiccator containing discolored silica gel at room temperature. Set the drying time to 30 min, and the water content is 12%;

[0010] (3) Freezing procedure: Place the sporangia dried for 30 min in step (2) into the LS loading solution and treat at 25 °C for 25 min;

[0011] The components and ratios of the LS loading solution are as follows: MS liquid culture medium containing 30% sucrose and 40% glycerol, sterilized by high-pressure steam at 121 °C for 20 min, cooled, and refrigerated at 4 °C;

[0012] (4) Vitrification treatment: After the treatment with the LS loading solution is completed, take out the sporangia and transfer them into a cryopreservation tube containing the PVS solution. Treat at 4 °C for 30 min, then replace it with the PVS2 solution pre-cooled at 4 °C and treat at 4 °C for 10 min;

[0013] The components and ratios of the PVS solution are as follows: 4.12 g / L MS + 30% glycerol + 15% ethylene glycol + 12% DMSO + 30% sucrose + 50 μmol / L serine / cysteine protease inhibitor + 0.5 g / L glutathione;

[0014] (5) Ultra-low temperature preservation: Immediately plunge the cryopreservation tube containing the sporangia treated in step (4) with the PVS2 solution into liquid nitrogen for ultra-low temperature preservation;

[0015] (6) Thawing and unloading treatment: Take out the cryopreservation tube containing the sporangia from liquid nitrogen, immediately place it in a 37 °C water bath for rapid thawing for 2 min, then place it on a clean bench, soak it 3 times with the US unloading solution for 5 min each time, and then wash it 3 times with sterilized pure water;

[0016] The components and ratios of the unloading solution are as follows: 4.12 g / L MS + 1.2 M sucrose.

[0017] Advantages of the present invention:

[0018] The present invention has successfully explored the vitrification ultra-low temperature preservation method of Huperzia serrata for the first time, and has explored the adjustment of the ultra-low temperature preservation protectant and the optimization of the key steps for the sporangia of Huperzia serrata. Finally, a vitrification ultra-low temperature preservation method for improving the survival rate of Huperzia serrata is provided. This method has a simple process, reliable stability, and the viability of the sporangia after preservation can reach more than 44.44%. The present invention can effectively and safely preserve Huperzia serrata for a long time, and is a practical new method for the long-term preservation of the germplasm resources of Huperzia serrata. Description of the Drawings

[0019] Figure 1 It is a Huperzia serrata plant. Detailed implementation manners

[0020] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0021] Example 1: Vitrification cryopreservation of Lycopodium serratum

[0022] (1) Material acquisition: Select plump, uniform, and healthy sporangia, and use the bromothymol blue method to measure the initial viability of the sporangia. Select sporangia with an initial viability ≥ 90% as the preservation materials;

[0023] (2) Screening for appropriate water content treatment: Place the sporangia selected in step (1) into a 2 mL centrifuge tube, open the lid of the centrifuge tube, and place the centrifuge tube in a desiccator containing discolored silica gel at room temperature. Set the drying time to 30 min, and the water content is 12%;

[0024] (3) Freezing procedure: Place the sporangia dried for 30 min in step (2) into the LS loading solution and treat at 25 °C for 25 min;

[0025] The components and ratios of the LS loading solution are as follows: MS liquid culture medium containing 30% sucrose and 40% glycerol, sterilized by high-pressure steam at 121 °C for 20 min, cooled, and refrigerated at 4 °C;

[0026] (4) Vitrification treatment: After the treatment with the LS loading solution is completed, take out the sporangia and transfer them into a cryopreservation tube containing the PVS solution, treat at 4 °C for 30 min, and then replace it with the PVS2 solution pre-cooled at 4 °C and treat at 4 °C for 10 min;

[0027] The components and ratios of the PVS solution are as follows: 4.12 g / L MS + 30% glycerol + 15% ethylene glycol + 12% DMSO + 30% sucrose + 50 μmol / L serine / cysteine protease inhibitor + 0.5 g / L glutathione;

[0028] (5) Cryopreservation: Immediately plunge the cryopreservation tube containing the sporangia treated in step (4) with the PVS2 solution into liquid nitrogen for cryopreservation;

[0029] (6) Thawing and unloading treatment: Take out the cryopreservation tube containing the sporangia from liquid nitrogen, immediately place it in a 37 °C water bath for rapid thawing for 2 min, then place it on a clean bench, soak it 3 times with the US unloading solution, 5 min each time, and then wash it 3 times with sterilized pure water;

[0030] The components and ratio of the unloading liquid are as follows: 4.12 g / L MS + 1.2 M sucrose.

[0031] Statistical detection of the survival rate after recovery culture of Huperzia serrata cryopreserved by the above method: Take the thawed sporangia, and use the bromothymol blue (BTB) method to determine the sporangium viability. The survival rate = (the number of sporangia with viability after recovery culture / the number of cryopreserved sporangia) × 100%, and the survival rate reaches 44.44%.

[0032] The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for cryopreservation of Huperzia serrata in vitrification, characterized in that, Using the sporangia of Huperzia serrata as preservation materials, the specific steps are as follows: (1) Material acquisition: Select plump, uniform, and healthy sporangia. Use the bromothymol blue method to determine the initial viability of the sporangia, and select sporangia with an initial viability ≥ 90% as preservation materials; (2) Screening for appropriate water content treatment: Place the sporangia selected in step (1) into a 2 mL centrifuge tube, open the lid of the centrifuge tube, and place the centrifuge tube in a desiccator containing discolored silica gel at room temperature. Set the drying time to 30 min, and the water content is 12%; (3) Freezing procedure: Place the sporangia dried for 30 min in step (2) into LS loading solution and treat at 25 °C for 25 min; The components and ratios of the LS loading solution are as follows: MS liquid culture medium containing 30% sucrose and 40% glycerol, autoclaved at 121 °C for 20 min, cooled, and refrigerated at 4 °C; (4) Vitrification treatment: After the treatment with LS loading solution is completed, take out the sporangia and transfer them into a cryopreservation tube containing PVS solution, treat at 4 °C for 30 min, and then replace it with pre-cooled PVS2 solution at 4 °C and treat at 4 °C for 10 min; (4) The components and ratios of the PVS solution are as follows: 4.12 g / L MS + 30% glycerol + 15% ethylene glycol + 12% DMSO + 30% sucrose + 50 μmol / L serine / cysteine protease inhibitor + 0.5 g / L glutathione; (5) Ultra-low temperature preservation: Quickly put the cryopreservation tube containing the sporangia with PVS2 solution after the treatment in step (4) into liquid nitrogen for ultra-low temperature preservation; (6) Thawing and unloading treatment: Take out the cryopreservation tube containing the sporangia from liquid nitrogen, immediately put it into a 37 °C water bath for rapid thawing for 2 min, then place it on a clean bench, soak it in US unloading solution 3 times, 5 min each time, and then wash it 3 times with sterilized pure water; (6) The components and ratios of the unloading solution are as follows: 4.12 g / L MS + 1.2 M sucrose.

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