Studies On The Protoplast Fusant With Hypsizigus Marmoreus (Peck) Bigelw And Flammulina Velutiper(Fr.)Sing.

Hypsizigus marmoreus (Peck) Bigelw is known as spot Hypsizigus marmoreus (Peck) Bigelw. It is also called crab mushroom, because it has crab taste. It is a kind of lumber saprophytic bacteria. It distributes in Europe, North America, Japan and Siberia. Hypsizigus marmoreus (Peck) Bigelw has many high nutritional materials, such as amino acid, vitamin, mineral element and polysaccharide. Hypsizigus marmoreus (Peck) Bigelw of the best quality and tasted well is a kind of health food which contain high protein, low fat and low calorie.Flammulina velutiper(Fr.)Sing. is called MaoBingjinqian bacteria. Flammulina velutiper(Fr.)Sing. is a kind of lumber saprophytic bacteria and distributes widely. Flammulina velutiper(Fr.)Sing. can promote children’s intelligence development, so it is called "zengzhi mushroom", "ikkyu mushroom". It also has the function of antineoplastic, immunoregulation, cholesterol lowing and hepatocyte protection, is a kind of vegetable and health food which contain rich vitamin, mineral element, high protein, low fat and low calorie.The test is aimed at get fusion strains with two parents complementary features. It lays the foundation for liquid fermentation, the extraction of active substances, fusion strains breeding research and get sporocarp which have commodity value by artificial cultivation.Test studies fluid medium of Hypsizigus marmoreus (Peck) Bigelw and Flammulina velutiper(Fr.)Sing., protoplast preparation and regeneration, inactivated tag, fusion conditions optimization and fusion strains appraisal by single factor test and the orthogonal test. The results are as follows:The optimum fluid medium of mycelium composition of Hypsizigusmarmoreus (Peck) Bigelw are maltose 3.00%, sucrose 3.00%, yeast extract 0.30%, wheat bran 1.00%, KH2PO40.10%, MgSO4·7H2O 0.05%,VB1 0.0009%, natural pH.The optimum fluid medium of mycelium composition of Flammulina velutiper(Fr.)Sing. are potato 15.00%, corn flour 3.00%, KH2PO40.02%, MgSO4·7H2O 0.05%, K2HPO40.08%。The optimum protoplast preparation condition of Hypsizigusmarmoreus (Peck) Bigelw are enzyme system 2.00%L, enzymolysis temperature 27℃, enzymolysis time 2.5h, osmotic stabilizer KCl(0.50mol/L), fungus age 4d.The optimum protoplast regeneration condition of Hypsizigusmarmoreus (Peck) Bigelw are enzyme system 2.00%L, enzymolysis temperature 25℃, enzymolysis time 3h, osmotic stabilizer MgSO4·7H2O (0.50mol/L), fungus age 5d.The optimum protoplast preparation condition of Flammulina velutiper(Fr.)Sing. are enzyme system 2.00%L, enzymolysis temperature 27℃, enzymolysis time 3h, osmotic stabilizer KCl(0.60mol/L), fungus age 7d.The optimum protoplast regeneration condition of Flammulina velutiper(Fr.)Sing. are enzyme system 1.00%L+1.00%C, enzymolysis temperature 27℃, enzymolysis time 2.5h, osmotic stabilizer MgSO4·7H2O (0.60mol/L), fungus age 7d.The optimum UV inactivation condition of Hypsizigusmarmoreus (Peck) Bigelw are:below the 15W UV-lamp,15cm away vertically irradiated for 50 seconds.The optimum heat inactivation condition of Flammulina velutiper(Fr.)Sing. 50℃ heat water bath treatment 40 minutes.The optimum protoplast fusion conditions of two parents are:using the mixture of 30.00% PEG6000 and 50 mmol/L Ca2+ to promote fusion, at 30℃ for 30 minutes pH8.5.The test authenticates fusion strains through rivalry experiment, colony feature comparison and mycelium morphology comparison, the mycelium growth rate between parents and fusion strains, and RAPD, ERIC molecular marker. The results show that fusant R1, R2 have close relataves with Hypsizigusmarmoreus (Peck) Bigelw. Fusant R3 has close relataves with Flammulina velutiper(Fr.)Sing. The all fusants come from the parents’fusion, and have the parents’features.