Study On Mononuclear Hybridization And Protoplast Fusion Quality Breeding Of Hericium Erinaceus

Hericium erinaceus is an edible and medicinal fungi belonging to Basidiomycota,we have a saying"Hericium delicacies,seafood bird's nest".The chemical ingredients of H.erinaceu is quite complex,which contains compounds such as polysaccharide,sterols,terpene,aliphatic acid and phenols.H.erinaceus fruit body also has various functions such as improving immunity,anti-oxidation,anti-tumor,hypolipidemic,anti-aging and so on.With the in-depth study of H.erinaceus,H.erinaceus products are also increasing,and the demand for H.erinaceus market is rapidly increasing.H.erinaceus is a prerequisite and basis for obtaining high-quality H.erinaceus.Therefore,breeding strains with high active metabolite content and high biological conversion rate have practical application value.In this article,the H.erinaceus strain 0605 with a higher bioconversion rate and Cichang strain with a better fruit body morphology were used as parent strains to prepare protoplast mononuclear bodies for mononuclear cross and protoplast fusion breeding.The resulting heterozygotes and fusions were obtained.Substances were identified,screened,and cultivated,and active hericium,nutrients,and appearance quality were used as main indicators to screen for the H.erinaceus species.This thesis consists main achievements are as follows:1.By performing single factor experiment and orthogonal experiment,it is confirmed that the best condition of preparation and regeneration of H.erinaceus protoplast is:enzyme system 2.0%lywallzyme+0.5%driselase,enzymolysis temparature 32?,enzymolysis duration 2.5h,cell age of mycelium 10d,constant temperature shaker rotate speed 120rpm,under such preparation condition,the quantity and regeneration rate of H.erinaceus protoplast is higher with 13.10×10⁶?/mL?0.83%respectively,H.erinaceus protoplast with higher quality could be obtained.2.In this study,18 strains of H.erinaceus 0605 monokaryons and 12 strains of H.erinaceus Cichang monokaryons were obtained through protoplast mononuclear hybridization,and 216 crosses were obtained from this material.Microscopic examination of both parental mononuclear bodies was performed.Whether or not the staggered mycelium had a lock-like combination resulted in 30 strains of H.erinaceus hybrid strains.Based on the analysis of the biological characteristics of 30 strains of H.erinaceus heterozygous strains and their parents,the mycelial growth rate of hybrid strain ZJ513increased by 45.46%relative to parent strain 0605,and the biomass of hybrid strain ZJ917increased by 43.08%relative to parent strain 0605.Analysis of the agronomic traits of 30strains showed that the highest yield of the hybrid strain ZJ1210 was increased by 7.14%compared to that of the parent strain;the fruit-body mushroom shape of the three strains of ZJ105,ZJ909 and ZJ1018 was better than that of both parents;The color of the fruiting bodies of strains ZJ903,ZJ906,ZJ908,ZJ909,ZJ910,ZJ917,ZJ1015,ZJ1018,ZJ1113,ZJ1204,ZJ1210,ZJ1211 was relatively white compared to that of the parent strain.3.The analysis of nutrient and active components of 30 fruiting bodies of H.erinaceus hybrid strains showed that ZJ917 had the highest polysaccharide content in fruiting bodies and 63.70%relative to parental 0605;the highest protein content in fruiting bodies was ZJ507,relative parent 0605 increased by 5.7%;the ergosterol content in the ZJ917 fruiting body was the highest,and the relative parental 0605 increased by 9.0%.The results of the taste substance analysis showed that the ZJ1113 fruit body had the highest flavor amino acid content and the ZJ1204 sweet taste amino acid was the highest,the umami taste nucleotide was the highest in the hybrid strain ZJ908,and the ZJ906 had the highest EUC value and reached the third level of the Mau grade.4.Optimization of parental inactivation conditions of protoplast fusion.The results showed that H.erinaceus 0605 protoplast was heat inactivated and treated at 55°C for 120mins.H.erinaceus Cichang protoplast was inactivated with ultraviolet light and the distance from a 15 W UV lamp was 20 cm.Irradiated for 26 s,the mortality rate reached100%.The optimal conditions for fusion of H.erinaceus 0605 and H.erinaceus Cichang with a single factor experiment were:30%PEG 6000,Ca²⁺concentration 10 mmol/L,fusion time 25 min,fusion temperature 32°C,With the fusion of pH 9.5,the maximum fusion rate was 2.499×10^-5%under this condition.5.67 strains of fusion strains were screened by SSCP analysis after selection of the fused strains.The biological characteristics of the fusion strains were identified as follows:The mycelial growth rates of the fusion strains R51'and R53'were the highest.It was 0.422cm/d faster,relative to the parent strain 0605,increased by 24.48%;R52'biomass was the highest,reaching 13.925 g/L,exceeding the parent's spiny biomass of 28.05%;mycelia polysaccharide content of the fusion strain R17'higher than the parent 0605,an increase of7.43%relative to the parent 0605.6.The agronomic traits analysis of the fruiting bodies of 14 H.erinaceus fusion strains showed that the fruit body morphologies of the fusion strains R29,R117,R19',and R52'were more regular than those of the parent strains,and the color of the fruit bodies was also whiter.The yield of R28 fruiting body was slightly higher than that of the parent strain at 146.39 g.The result of polysaccharide content of fruiting body showed that the content of polysaccharides of two fusion strains R29 and R52'in 14 fusion strains was higher than that of the parent 0605,which increased by 22.27%and 22.78%respectively.The results of free amino acid content in fruiting bodies showed that the glutamic acid content,umami taste amino acid content and sweet taste amino acid content of the fusion strain R29 in the 14 fusion strains were the highest and were 9.26 mg/g,12.09 mg/g,and5.90 mg/g,respectively,excluding the parent strain 0605 5.47%,6.70%and 9.46%.