Protoplast fusion method of macrocybe gigantea (Massee)Pegler&Lodge and agaricus bisporus
A technology of protoplast fusion and giant trichomonas, which is applied in the field of protoplast fusion of gigantic Tricholoma gigantea and Agaricus bisporus, to achieve the effects of benefiting food safety, improving protoplast fusion rate, and strong repeatability
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Embodiment 1
[0073] (1) Mycelium preparation:
[0074] On the PDA medium, take 2 to 3 pieces of giant Tricholoma mycelium pieces that grow vigorously at the front, inoculate them into a 250mL Erlenmeyer flask filled with 160mL liquid PDA medium, and cultivate them in a shaker at 26°C and 160r / min On 7-10 days, take an appropriate amount of cultured mycelium balls and transfer them to a 250mL Erlenmeyer flask filled with 160mL of liquid PDA medium, and culture them at 30°C for more than 4 days, pick mycelia under sterile conditions, and blot them dry with sterile filter paper Moisture, get the fresh mycelium of Tricholoma gigantea.
[0075] On the PDA medium, take 2 to 3 slices of Agaricus bisporus mycelium pieces that are vigorously growing at the front, inoculate them into a 250mL Erlenmeyer flask filled with 160mL liquid PDA medium, and place them in a shaker at 26°C and 160r / min Cultivate for 7-10 days, transfer an appropriate amount of cultured mycelia balls to a 250-mL Erlenmeyer fla...
Embodiment 2
[0110] (1) Fermentation cultivation materials: take cultivation raw materials (bagasse, cottonseed hulls, bran, KH 2 PO 4 , CaCO 3 ), add water and mix well, fully stir with a mixer during the process of adding water to make it fully absorb water, then add an appropriate amount of lime water and mix well to make the pH value 7.0-9.0, build a heap and ferment for 7-10 days.
[0111] (2) Bagging: Adjust the water content of the fermented cultivation material to 58% to 68%, pH 7.0 to 9.0, put it into 22×44 polypropylene plastic bags, each bag weighs 1500±10g, and compact it to make the tightness Consistently, punch a hole in the middle to 1 / 5 of the bottom of the bag, then close tightly with a collar and a plastic lid with a sponge to filter the air.
[0112] (3) Sterilization: Sterilize at 121°C for 4-6 hours by high-pressure sterilization method, until it reaches a sterile state, move it into the cooling room, and cool the material temperature to about 30°C.
[0113] (4) Ino...
Embodiment 3
[0128] In step (5) of Example 1, during the fusion process of Tricholoma gigantea and Agaricus bisporus protoplasts, select PEG with a molecular weight of 6000 (concentrations are respectively 10%, 20%, 30%, 40%, and 50% of the mass-volume ratio), Ca 2+ Concentration of 0.01mol / L, 10mmoL / L Tris-HCl buffer solution with pH value of 8.0, PEG fusion treatment at 30°C for 20min. The result is as Figure 9 As shown, when the concentration of PEG6000 was 30%, the fusion rate of Tricholoma giantis and Agaricus bisporus protoplasts was the highest, reaching 1.07×10 -4 .
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