Method for breeding tremella aurantialba strains by using protoplast regeneration technology

Abstract

The invention discloses a method for breeding a tremella aurantialba strain by using a protoplast regeneration technology, and relates to the technical field of edible mushroom planting, the method for breeding the tremella aurantialba strain by using the protoplast regeneration technology comprises the following steps: respectively preparing protoplast of stereum hirsutum and protoplast of common tremella aurantialba, inducing fusion of the protoplast by using a reagent, a fusion strain is generated through a cell wall regeneration technology, a needed novel strain is selected through an artificial planting and screening mode, the problems that in existing tremella aurantialba planting, a strain source is fixed, stress resistance is degraded, and production performance is low are solved, tremella aurantialba parasitizing stereum hirsutum in the nature is re-engraved, and the advantages of the tremella aurantialba and stereum hirsutum are achieved; and the generation of the initial tremella aurantialba strain is quickly recarved in a mode of combining induced variation with manual screening, so that the stable high yield of the commodity tremella aurantialba planting strain is ensured.

Description

Technical Field

[0001] The invention relates to a protoplast regeneration technology and an edible fungus planting technology, and in particular to a method for breeding golden ear strains using a protoplast regeneration technology. Background Art

[0002] Golden ear is an edible fungus that parasitizes on the growth of Acanthopanax pubescens. The artificially cultivated strains in the existing technology are separated from the fruiting bodies of wild Golden ear, and need to be continuously reproduced through the fruiting bodies to provide strains, resulting in a small source of seed sources and the degradation of strain production performance, stress resistance, etc.

[0003] The quality of commercial golden ear fungus strains in the existing environment is uneven, and with the degradation of the production performance of the strains, the yield is reduced and the cultivation cost is high. There is a lack of stable, high-yield and stress-resistant strains. The use of existing commercial golden ear strains for cultivation cannot guarantee product quality and yield, and cannot adapt to the requirements of modern agricultural refined management and high-efficiency output. In order to promote the upgrading and development of the golden ear cultivation industry, it is urgently necessary to select and breed new golden ear varieties that are stable, high-yield and disease-resistant.

[0004] To this end, technicians in this field use the method of host and parasite protoplast infection and hybridization to induce hybridization, combined with artificial breeding to obtain more economically efficient Tremella fuciformis strains to meet the needs of commercial cultivation. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for breeding golden ear strains using protoplast regeneration technology, which combines the advantages of golden ear fungus and golden ear by inducing protoplast fusion, and combines artificial screening to obtain high-yield and short-cycle golden ear strains, thereby enhancing the stress resistance of stable cultivated golden ear strains and increasing the cultivation value.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: a method for breeding Tremella fuciformis strains by protoplast regeneration technology, comprising the following steps:

[0007] S1. Protoplasts of the fruiting bodies of Psoralea corylifolia and Psoralea corylifolia are prepared respectively, and the two protoplasts are mixed in equal proportions to obtain mixed protoplasts;

[0008] S2, diluting the mixed protoplasts with a nutrient solution, and then adding an induction agent to induce the protoplasts to fuse;

[0009] S3, diluting the washing induction solution, extracting the protoplasts by centrifugation to regenerate the cell wall, obtaining hybrid strains, and culturing the hybrid strains with a nutrient medium to obtain strain sol;

[0010] S4, boiling the culture medium at high temperature, cooling it to room temperature, bagging and compacting it, planting the strains on one side and multiple points on the culture medium with a syringe, cultivating the proliferating hyphae, and recording the growth status of the hyphae at regular intervals;

[0011] S5. Open the bag at the position where the mycelium grows well to induce the growth of golden ear fruiting bodies, number the golden ear fruiting bodies at different positions and take samples for seed preservation, and then record the growth status of the golden ear fruiting bodies;

[0012] S6. Analyze the growth of golden ear fruiting bodies with different numbers by data, and select numbered samples with high yield and short growth cycle for breeding and cultivation.

[0013] Obtain target protoplasts:

[0014] 1. Materials

[0015] General Tremella species, Lederma lucidum species, Lywallzyme (Lywallzyme, Institute of Microbiology, Guangdong Academy of Sciences, Guangdong Microbial Culture Collection Center), Sucrose (Tianjin Best Chemical Co., Ltd.), Magnesium sulfate (MgSO4, Shanghai McLean Biochemical Technology Co., Ltd.), Potassium dihydrogen phosphate (KH2PO4, Tianjin Zhiyuan Chemical Reagent Co., Ltd.), Maltose (C 12 H 22 O 11 .H2O (Tianjin Komiou Chemical Reagent Co., Ltd.), glucose (CHCHO), yeast extract powder, mannitol, agar, pure water, pipettes, several corresponding pipette tips, and several 2 ml pipettes.

[0016] 2. Instruments

[0017] Sterile workbench (Suzhou Antai Air Technology Co., Ltd.), vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), water purifier;

[0018] 3. Experimental steps

[0019] 1. Prepare mycelium and sucrose solution;

[0020] 2. Secondly, prepare enzyme solution and rejuvenation medium;

[0021] 3. Prepare protoplasts;

[0022] 4. Dilute and apply;

[0023] 5. Pick out suitable single-cell colonies on the regeneration medium and place them on the Tremella fuciformis medium.

[0024] 4. The detailed steps are as follows

[0025] 1.1 Preparation of common golden ear liquid culture medium

[0026] Formula: 10g corn flour, 3g peptone and potassium dihydrogen phosphate (KH2PO4), 1.5g magnesium sulfate (MgSO4), 1 liter water;

[0027] 1.2 Place the mycelium on the plate into a culture dish with filter paper, chop it into pieces, and then let it suspend on the surface of the liquid culture medium to grow bacterial balls.

[0028] 1.3 Prepare sucrose solution 0.6mol / L×0.2L=0.12mol

[0029] Sucrose mass 0.12*342.29=41.0g;

[0030] 2.1 Enzyme solution 2% lytic enzyme (if 4 ml is prepared)

[0031] Weigh 0.04 g of lytic enzyme using a precision balance and place it in a sterilized centrifuge tube. Add 4 ml of sucrose solution and sterilize it using a 0.22 μm filter.

[0032] 2.2 Solid regeneration medium: 10 g maltose, 5 g glucose, 5 g yeast extract powder, 109.2 g mannitol (0.6 mol / L), 15 g agar, distilled water to 1 L;

[0033] 3.1 Protoplast preparation: After picking the mycelium into a centrifuge tube, centrifuge to separate the mycelium from the culture medium, centrifuge with the corresponding osmotic stabilizer at 6000r / min for 10min, wash once, and remove excess water with sterile filter paper. Add 1ml enzyme solution for every 100mg of mycelium and place in a constant temperature water bath at 25-35℃ for 2-3h;

[0034] Protoplast purification: After the corresponding enzymatic hydrolysis time, the filtrate is centrifuged at 3500r / min for 10min, the supernatant is discarded, the precipitate is washed twice by centrifugation with the corresponding osmotic stabilizer, and the protoplasts are resuspended in the osmotic stabilizer.

[0035] 4.1 Dilution: Pipette 100 μl of the liquid with protoplasts into a 2 ml pipette and add 900 μl of sucrose solution. Repeat this step five times. If the dilution factor is 10-4 to 10-5, take 200 μl of the diluted solution and inject it into the regeneration plate and spread it evenly with a spreader. If the dilution factor is ten to the power of -3, inject 100 μl of the diluted solution into the regeneration puzzle plate and spread it evenly.

[0036] 5. Cultivation

[0037] Place the coated plates at 20°C for incubation;

[0038] 6. Pick out the colonies.

[0039] Protoplast induced fusion

[0040] The prepared Tremella fusca protoplasts are mixed with the protoplasts of Lederma lucidum, and diluted with a liquid nutrient medium, a high concentration (30%-50%) PEG solution is added to the mixed protoplast solution, and the solution is treated at 20-25° C. for 10-30 minutes, and the PEG is slowly diluted with a diluent containing calcium and magnesium ions, the diluted solution is centrifuged, and the fused protoplasts are extracted for cell wall regeneration to obtain a mixed hybrid strain.

[0041] Planting and manual selection

[0042] Preparation of nutrient medium

[0043] One or more of sawdust from broad-leaved trees such as poplar, willow, and oak, wheat bran, rice husks, corn cobs, cottonseed hulls, and straw are crushed and mixed with quicklime, stirred and fermented with water, bagged and compacted, and then sterilized by high-temperature steaming and naturally cooled to room temperature to obtain a planting base.

[0044] Planting and manual selection

[0045] The hybrid liquid strains are injected into the planting base at equal intervals using a syringe, and the mycelium is cultivated under appropriate temperature and humidity. After the mycelium covers the entire nutrient base, a bag is opened at an appropriate position to induce the growth of the golden ear fruiting body;

[0046] Wait for the fruiting body to grow 10-20mm, take samples from the fruiting body, number the sampled strains, save the samples, continue to culture the fruiting body, and record the growth of the fruiting body every other day;

[0047] Wait for the fruiting body to grow to 120dm 3 The fruiting bodies are harvested and dried, and the dried Tremella fuciformis products are weighed and recorded;

[0048] The desired strain is selected based on the growth rate of the mycelium, the growth rate of the fruiting body and the dry weight of the mature fruiting body, and the corresponding numbered seeds are taken for artificial breeding.

[0049] The present invention discloses a method for breeding Tremella fuciformis strains by using protoplast regeneration technology, which has the following beneficial effects:

[0050] The method for breeding golden ear strains using the protoplast regeneration technology comprises the following steps: preparing protoplasts of hairy leathery fungus and common golden ear respectively, inducing fusion of the protoplasts using reagents, generating fused strains using cell wall regeneration technology, and selecting required new strains through artificial cultivation and screening to improve the existing golden ear cultivation strains, such as fixed source, deterioration of stress resistance, and low production performance. The method replicates the hairy leathery fungus parasitic on golden ear in nature, possessing the advantages of both, and rapidly replicating the production of initial golden ear strains by inducing mutations combined with artificial screening to ensure stable and high yields of commercial golden ear cultivation strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1-3 A plate diagram of the protoplasts regenerated from the 905 leather fungus prepared by the present invention;

[0053] Figure 4-6 This is a plate diagram of the Tremella truncatula regeneration protoplasts prepared in the present invention. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The embodiment of the present invention discloses a method for breeding Tremella fuciformis strains using protoplast regeneration technology;

[0056] The steps include:

[0057] S1. Protoplasts of the fruiting bodies of Psoralea corylifolia and Psoralea corylifolia are prepared respectively, and the two protoplasts are mixed in equal proportions to obtain mixed protoplasts;

[0058] S2, diluting the mixed protoplasts with a nutrient solution, and then adding an induction agent to induce the protoplasts to fuse;

[0059] S3, diluting the washing induction solution, extracting the protoplasts by centrifugation to regenerate the cell wall, obtaining hybrid strains, and culturing the hybrid strains with a nutrient medium to obtain strain sol;

[0060] S4, boiling the culture medium at high temperature, cooling it to room temperature, bagging and compacting it, planting the strains on one side and multiple points on the culture medium with a syringe, cultivating the proliferating hyphae, and recording the growth status of the hyphae at regular intervals;

[0061] S5. Open the bag at the position where the mycelium grows well to induce the growth of golden ear fruiting bodies, number the golden ear fruiting bodies at different positions and take samples for seed preservation, and then record the growth status of the golden ear fruiting bodies;

[0062] S6. Analyze the growth of golden ear fruiting bodies with different numbers by data, and select numbered samples with high yield and short growth cycle for breeding and cultivation.

[0063] Steps and preparations for preparing protoplasts of fungi:

[0064] 1. Materials

[0065] Lederma strains, Lywallzyme (Lywallzyme, Institute of Microbiology, Guangdong Academy of Sciences, Guangdong Microbial Culture Collection Center), sucrose (Tianjin Best Chemical Co., Ltd.), magnesium sulfate (MgSO4, Shanghai McLean Biochemical Technology Co., Ltd.), potassium dihydrogen phosphate (KH2PO4, Tianjin Zhiyuan Chemical Reagent Co., Ltd.), maltose (C 12 H 22 O 11 .H2O Tianjin Kemeiou Chemical Reagent Co., Ltd.), glucose (CHCHO) n , yeast extract powder, mannitol, agar, pure water, pipettes, several corresponding pipette tips, and several 2 ml pipettes.

[0066] 2. Instruments

[0067] Sterile workbench (Suzhou Antai Air Technology Co., Ltd.), vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), water purifier.

[0068] 3. Experimental steps

[0069] 1. Prepare mycelium and sucrose solution;

[0070] 2. Secondly, prepare enzyme solution and rejuvenation medium;

[0071] 3. Prepare protoplasts;

[0072] 4. Dilute and apply;

[0073] 5. Pick out suitable single-cell colonies on the regeneration medium and place them on the Tremella fuciformis medium.

[0074] 4. The detailed steps are as follows

[0075] 1.1 Preparation of common golden ear liquid culture medium

[0076] Formula: 10g corn flour, 3g peptone and potassium dihydrogen phosphate (KH2PO4), 1.5g magnesium sulfate (MgSO4), 1 liter water;

[0077] 1.2 Place the mycelium on the plate into a culture dish with filter paper, chop it into small pieces, and then let it suspend on the surface of the liquid culture medium to grow bacterial balls;

[0078] 1.3 Prepare 0.6 mol / L sucrose solution

[0079] Sucrose mass 0.6mol / L×0.2L=0.12mol

[0080] 0.12*342.29=41.0g.

[0081] 2.1 Enzyme solution 2% lytic enzyme (if prepared 2 ml)

[0082] Weigh 0.04 g of lytic enzyme using a precision balance and place it in a sterilized centrifuge tube. Add 4 ml of sucrose solution and sterilize it using a 0.22 μm filter.

[0083] 2.2 Solid regeneration medium: maltose 10g, glucose 5g, yeast extract 5g, mannitol 109.2g (0.6mol / L), agar 15g, distilled water to 1L.

[0084] 3.1 Protoplast preparation: After picking the mycelium into a centrifuge tube, centrifuge to separate the mycelium and the culture medium, centrifuge with the corresponding osmotic stabilizer at 6000r / min for 10min, wash once, and absorb excess water with sterile filter paper;

[0085] In a 2 ml centrifuge tube containing two 2.5 mm diameter glass beads, add 1 ml of enzyme solution for every 100 mg of mycelium, shake thoroughly until the mycelium is broken up, and place in a constant temperature water bath at 25-35°C for 2-3 hours;

[0086] Protoplast purification: After the corresponding enzymatic hydrolysis time, the protoplast suspension was filtered through an 18 μm sterile filter to remove residual mycelial fragments. The filtrate was centrifuged at 3500 r / min for 10 min, the supernatant was discarded, the precipitate was washed twice by centrifugation with the corresponding osmotic stabilizer, and the protoplasts were resuspended in the osmotic stabilizer.

[0087] 4.1 Dilution: Pipette 100 μl of the liquid containing protoplasts into a 2 ml pipette and add 900 μl of sucrose solution. Repeat this step three times, then take 100 μl of the diluted solution and inject it into the regeneration plate and spread it evenly with a spreader.

[0088] 5. Cultivation

[0089] The coated plates were incubated at 20°C.

[0090] 6. Pick out the colonies.

[0091] Steps and preparations for preparing Tremella fusca protoplasts.

[0092] 1. Materials

[0093] Tremella fuciformis, Lywallzyme (Lywallzyme, Institute of Microbiology, Guangdong Academy of Sciences, Guangdong Microbial Culture Collection Center), Sucrose (Tianjin Best Chemical Co., Ltd.), Magnesium sulfate (MgSO4, Shanghai McLean Biochemical Technology Co., Ltd.), Potassium dihydrogen phosphate (KH2PO4, Tianjin Zhiyuan Chemical Reagent Co., Ltd.), Maltose (C 12 H 22 O 11 .H2O (Tianjin Komiou Chemical Reagent Co., Ltd.), glucose (CHCHO)n, yeast extract powder, mannitol, agar, pure water, pipettes, several corresponding pipette tips, and several 2 ml pipettes.

[0094] 2. Instruments

[0095] Sterile workbench (Suzhou Antai Air Technology Co., Ltd.), vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), water purifier.

[0096] 3. Experimental steps

[0097] 1. Prepare mycelium and sucrose solution;

[0098] 2. Secondly, prepare enzyme solution and rejuvenation medium;

[0099] 3. Prepare protoplasts;

[0100] 4. Dilute and apply;

[0101] 5. Pick out suitable single-cell colonies on the regeneration medium and place them on the Tremella fuciformis medium.

[0102] 4. The detailed steps are as follows

[0103] 1.1 Preparation of common golden ear liquid culture medium

[0104] Recipe: 10g corn flour, 3g each of peptone and potassium dihydrogen phosphate (KH2PO4), 1.5g magnesium sulfate (MgSO4), and 1 liter of water.

[0105] 1.2 Place the mycelium on the plate into a culture dish with filter paper, chop it into pieces, and then let it suspend on the surface of the liquid culture medium to grow bacterial balls.

[0106] 1.30.6mol / L sucrose solution

[0107] Sucrose mass 0.6mol / L×0.2L=0.12mol

[0108] 0.12*342.29=41.0g.

[0109] 2.1 Enzyme solution 2% lytic enzyme (if 4 ml is prepared)

[0110] Weigh 0.04 g of lytic enzyme using a precision balance and place it in a sterilized centrifuge tube, add 4 ml of sucrose solution, and sterilize with a 0.22 μm filter.

[0111] 2.2 Solid regeneration medium: maltose 10g, glucose 5g, yeast extract 5g, mannitol 109.2g (0.6mol / L), agar 15g, distilled water to 1L.

[0112] 3.1 Protoplast preparation: After picking the mycelium into a centrifuge tube, centrifuge to separate the mycelium and the culture medium, centrifuge with the corresponding osmotic stabilizer at 6000r / min for 10min, wash once, and absorb excess water with sterile filter paper;

[0113] Add 1 ml of enzyme solution to every 100 mg of mycelium and place in a constant temperature water bath at 25-35°C for 2-3 hours;

[0114] Protoplast purification: After the corresponding enzymatic hydrolysis time, the filtrate is centrifuged at 3500r / min for 10min, the supernatant is discarded, the precipitate is centrifuged and washed twice with the corresponding osmotic stabilizer, and the protoplasts are resuspended in the osmotic stabilizer.

[0115] 4.1 Dilution: Pipette 100 μl of the liquid with protoplasts into a 2 ml pipette and add 900 μl of sucrose solution. Repeat this step five times. If the dilution factor is 10 -4 Up to 10 -5 Take 200 μl of the diluted solution, inject it into the regeneration plate and spread it evenly with a spreader;

[0116] If the dilution factor is 10 -5 , take 100 microliters of the diluted solution, inject it into the regenerated puzzle board and spread it evenly.

[0117] 5. Cultivation

[0118] Place the coated plates at 20°C for incubation.

[0119] 6. Pick out the colonies.

[0120] Protoplast induced fusion

[0121] The prepared Tremella fusca protoplasts are mixed with the protoplasts of Lederma lucidum, and diluted with a liquid nutrient medium, a high concentration (30%-50%) PEG solution is added to the mixed protoplast solution, and the solution is treated at 20-25° C. for 10-30 minutes, and the PEG is slowly diluted with a diluent containing calcium and magnesium ions, the diluted solution is centrifuged, and the fused protoplasts are extracted for cell wall regeneration to obtain a mixed hybrid strain.

[0122] Planting and manual selection

[0123] Preparation of nutrient medium

[0124] Use sawdust from broad-leaved trees such as poplar, willow, oak as the matrix, add one or more of wheat bran, rice husks, corn cobs, cottonseed hulls, and straw, crush them and mix with quicklime, add water, stir and ferment, bag and compact, then perform high-temperature steaming and sterilization, and naturally cool to room temperature to obtain a planting base.

[0125] Planting and manual selection

[0126] The hybrid liquid strains are injected into the planting base at equal intervals using a syringe, and the mycelium is cultivated under appropriate temperature and humidity. After the mycelium covers the entire nutrient base, a bag is opened at an appropriate position to induce the growth of the golden ear fruiting body;

[0127] Wait for the fruiting body to grow 10-20mm, take samples from the fruiting body, number the sampled strains, save the samples, continue to culture the fruiting body, and record the growth of the fruiting body every other day;

[0128] Wait for the fruiting body to grow to 120dm 3 The fruiting bodies are harvested and dried, and the dried Tremella fuciformis products are weighed and recorded;

[0129] The desired strain is selected based on the growth rate of the mycelium, the growth rate of the fruiting body and the dry weight of the mature fruiting body, and the corresponding numbered seeds are taken for artificial breeding.

[0130] The openings on the nutrient substrate are numbered, and the growth rate of the hyphae and the growth rate of the fruiting body are reversed to obtain a comparison table as follows:

[0131] 1 2 3 4 5 6 7 8 9 10 92 115 102 98 106 92 110 108 112 94 12(h) 0.15 0.13 0.21 0.18 0.19 0.16 0.17 0.15 0.18 0.21 24(h) 0.32 0.35 0.41 0.28 0.32 0.33 0.35 0.38 0.20 0.45 36(h) 0.86 0.95 1.12 0.96 0.88 1.05 1.08 0.97 1.07 1.22 48(h) 3.25 3.66 4.12 3.72 3.88 4.65 4.25 3.77 4.32 5.55 60(h) 9.21 9.95 10.8 9.82 9.99 10.5 9.88 9.76 11.2 14.6 72(h) 22.6 26.9 32.7 28.5 32.2 33.1 29.3 29.7 33.3 35.6 84(h) 47.7 58.4 69.8 56.2 66.5 73.8 54.6 61.2 69.8 77.5 96(h) 82.5 79.3 99.8 87.5 98.2 102 78.4 89.9 96.5 108 118(h) 107 100 120 115 120 120 108 111 120 120

[0132] The first column above is the time it takes to trace the mycelium of the strain to cover the entire planting base. The fruiting bodies are measured by volume. The fruiting bodies are harvested when they reach a volume of 120 cubic centimeters. The harvested strains are dried and their quality is compared. The dry weight of the strains is included in the number accordingly, which serves as an important reference factor for artificial breeding.

[0133] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for breeding Tremella fuciformis strains using protoplast regeneration technology, characterized in that: The steps include: S1. Protoplasts of the fruiting bodies of Psoralea corylifolia and Psoralea corylifolia are prepared respectively, and the two protoplasts are mixed in equal proportions to obtain mixed protoplasts; S2, diluting the mixed protoplasts with a nutrient solution, and then adding an induction agent to induce the protoplasts to fuse; S3, diluting the washing induction solution, extracting the protoplasts by centrifugation to regenerate the cell wall, obtaining hybrid strains, and culturing the hybrid strains with a nutrient medium to obtain strain sol; S4, boiling the culture medium at high temperature, cooling it to room temperature, bagging and compacting it, planting the strains on one side and multiple points on the culture medium with a syringe, cultivating the proliferating hyphae, and recording the growth status of the hyphae at regular intervals; S5. Open the bag at the position where the mycelium grows well to induce the growth of golden ear fruiting bodies, number the golden ear fruiting bodies at different positions and take samples for seed preservation, and then record the growth status of the golden ear fruiting bodies; S6. Analyze the growth of golden ear fruiting bodies with different numbers by data, and select numbered samples with high yield and short growth cycle for breeding and cultivation.

2. The method for breeding Tremella fuciformis strains using the protoplast regeneration technology as claimed in claim 1, characterized in that: Preparation of protoplasts of the fruiting bodies of Leymus tricholoma and Tremella ovata in step S1: ① Take corn flour, peptone, potassium dihydrogen phosphate and magnesium sulfate, add water and mix to make a common liquid culture medium, inoculate mycelium in the liquid culture medium for amplification culture; ② Mix maltose, glucose, yeast extract powder, mannitol and agar, dilute with distilled water to make a solid regeneration medium, and inoculate the bacterial balls amplified in the liquid medium into the solid regeneration medium for culture; ③ Hydrolyze the pellet with lytic enzyme, add sucrose solution to dilute, and then filter and sterilize the dilution to obtain a protoplast mixed solution; ④ Pipette the liquid containing protoplasts into a pipette, add sucrose solution to dilute, repeat this step three times to obtain the target protoplast solution.

3. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 2, characterized in that: The mycelium was picked up and placed in a centrifuge tube for centrifugation to separate the mycelium from the culture medium, an osmotic stabilizer was added, and the tube was centrifuged at 6000 r / min for 10 min, washed, and excess water was removed with sterile filter paper; In a 2 ml centrifuge tube containing two 2.5 mm diameter glass beads, add 1 ml of enzyme solution for every 100 mg of mycelium, shake thoroughly until the mycelium is broken up, and place in a constant temperature water bath at 25-35°C for 2-3 hours; After the corresponding enzymatic hydrolysis time, the protoplast suspension was filtered through an 18 μm sterile filter to remove residual mycelial fragments, centrifuged at 500 r / min for 10 min, the supernatant was discarded, the precipitate was centrifuged and washed twice with the corresponding osmotic stabilizer, and the protoplasts were resuspended in the osmotic stabilizer to complete the purification of the protoplasts.

4. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 2, characterized in that: Liquid culture medium: The mass ratio of corn flour, peptone, potassium dihydrogen phosphate, and magnesium sulfate is 10:3:3:1.5, and one liter of water is added for dilution.

5. The method for breeding Tremella fuciformis strains using the protoplast regeneration technology as claimed in claim 2, characterized in that: Solid regeneration medium: the mass ratio of maltose, glucose, yeast extract powder, mannitol, and agar is 10:5:5:109.2:15, the concentration of mannitol is 0.6 mol / L, and the volume is made up to 1 L with distilled water.

6. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 2, characterized in that: The concentration of the sucrose solution is 0.6 mol / L, the mass of sucrose is 0.12*342.29=41.0 g, and it is sterilized by filtration at 0.22 μm.

7. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 2, characterized in that: During the protoplast preparation operation, add 1 ml of lytic enzyme solution for every 100 mg of mycelium and place it in a constant temperature water bath at 25-35°C for 2-3 hours.

8. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 7, characterized in that: Protoplast purification: After the corresponding enzymatic hydrolysis time, the filtrate is centrifuged at 3500r / min for 10min, the supernatant is discarded, the precipitate is centrifuged and washed twice with the corresponding osmotic stabilizer, and the protoplasts are resuspended in the osmotic stabilizer.

9. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 7, characterized in that: Pipette 100 μl of the liquid with protoplasts into a 2 ml pipette and add 900 μl of sucrose solution. Repeat this step five times. If the dilution factor is 10 -4 Up to 10 -5 , take 200 microliters of the diluted solution, inject it into the regeneration plate and spread it evenly with a spreader. If the dilution factor is 10 -3 , take 100 μl of the diluted solution, inject it into the regeneration plate and spread it evenly.

10. The method for breeding Tremella fuciformis strains by protoplast regeneration technology as claimed in claim 1, characterized in that: In step S2, a high concentration (30%-50%) PEG solution is added to the mixed protoplast solution, and the solution is treated at 20-25° C. for 10-30 minutes. The PEG is then slowly diluted with a diluent containing calcium and magnesium ions. The diluted solution is centrifuged, and the fused protoplasts are extracted for cell wall regeneration to obtain a mixed hybrid strain.

Images