Breeding Of Agaricus Blazei High-Yield Vitamin B₁₂ Strains

Agaricus blazei has rich edible and medicinal value,considerable cultivation benefits,is conducive to resource recycling,and has broad development prospects.However,domestic germplasm resources are lacking,so it is necessary to conduct research on Agaricus blazei breeding.With the development of society,vegetarians are increasing,and vegetarians generally lack vitamin B₁₂.Accordingly,in this study,Agaricus Blazei Murill JS01 was used as the test material to obtain its protoplasts and spores respectively,and to obtain a high-yield vitamin B₁₂ Agaricus Blazei Murill strain through ultraviolet mutagenesis test,screening and identification of mutagenic strains.The results are as follows:1.Through single factor experiment and orthogonal experiment,using SPSS 22.0 software for analysis to optimize the preparation and regeneration conditions of Agaricus blazei.The results showed that the best conditions for the protoplast preparation were:7 days old,0.6mol·L^-1 KCl as osmotic pressure stabilizer,1.5%lysolytic enzyme temperature,30℃temperature,4.0 h,pH 6.0,Under these conditions,the protoplast yield was as high as 1.97×10⁷ cells·mL^-1;the bacteria age was 6 d,and 0.6 mol·L^-1 MgSO₄ was used as the osmotic pressure stabilizer.The lysozyme concentration was 2.0%,and the enzymolysis temperature was 30℃.When the decomposition time was 3.0 h,pH 6.5,and the regeneration medium was GM,the regeneration rate of protoplasts was the highest at 1.12%.After adding cellobiose and vitamin B₁ to the regeneration medium,the regeneration rate was 1.17%,which was 4.46%higher than the optimization result.2.Through the ultraviolet mutagenesis test,the screening and identification test of the mutagenic strains,the high-yield vitamin B₁₂ strains were bred.The results showed that the optimal time for UV mutagenesis of protoplasts and spores of Agaricus blazei was 55 s and 4min,respectively.Comparatively speaking,spores can obtain a large number of mutagenic strains in a short period of time due to low acquisition difficulty,easy storage and easy germination,which is more suitable for mutation breeding research.Through high-performance liquid detection,the content of vitamin B₁₂ in JS01 was 3.7954μg·g^-1,and the mutagenic strain BUV-16 was screened out.Its content was 4.1089μg·g^-1,an increase of 8.26%compared with JS01.Through ITS amplification and phylogenetic tree analysis,the results show that BUV-16,Agaricus blazei KF281111.1,Agaricus blazei MF403088.1,and JS01 have close evolutionary relationships;BUV-16 and Agaricus subrufescens KJ541802.1,Agaricus subrufescens KJ541804,Agaricus pseudominipurpureus MG196356.1,Agaricus rufoaurantiacus KT951558.1,Agaricus sp.EU284018.1 Evolutionary relationship is far away.3.Cultivation experiments were carried out on the selected mutant strains and their parents,and the content of vitamin B₁₂ in the fruiting bodies was separately detected.The results showed that the BUV-16 fruiting body mushroom type was normal,and its size,umbrella diameter,stalk length,etc.were not significantly different from the fruit body of Agaricus blazei JS01,according to statistics,the yield of Agaricus blazei JS01 was 4.40 kg·m^-2,and the yield of BUV-16 was 4.15 kg·m^-2.The difference in yield was small,indicating that BUV-16 could be used for normal cultivation.After high-performance liquid detection,the content of vitamin B₁₂ in the fruit body of Agaricus blazei JS01 was 3.9014μg·g^-1;the content of vitamin B₁₂ in the fruit body of BUV-16 was 4.1866μg·g^-1,which was 7.31%higher than that of JSO1 fruit body.Compared with the respective mycelium,the content of vitamin B₁₂ had increased to a certain extent,but the difference was small.The reason might be that the bacteria attached to the cap have a certain effect on its content,and the content of vitamin B₁₂ is different in different sampling locations.