Research On The Key Technology Of Factory Cultivation Of White Grifola Frondosa And Breeding Of New Varieties

Grifola frondosa is a rare dual-use mycorrhizal fungus.At present,the factory cultivation of G.frondosa in China is still in the preliminary exploration stage,and the strains in production are mostly imported from Japan or domesticated from wild dominant strains,which generally have the disadvantages of low yield,poor biotransformation rate and dark color.In this thesis,we used white G.frondosa Gr0001+3 as the test material and optimized the culture conditions of G.frondosa liquid strain by shaking flask liquid culture with mycelial biomass as the main index;judged the maturity of G.frondosa Gr0001+3 by observing and analyzing the changes of physicochemical indexes and key enzyme activities of the packs at different culture times and the relationship between culture time and mushroom yield;Exploring the molecular mechanisms regarding the developmental process of the cotyledons of ashwagandha by gene expression level analysis using RNA-Seq sequencing technology.The main findings are as follows:1.Orthogonal and response surface analysis were used to optimize the culture conditions of the liquid strain of golden mushroom of G.frondosa.The results showed that the best liquid strain medium culture formula was 28.5 g glucose,11.5 g yeast powder,carbon to nitrogen ratio10:1,and the mycelial biomass was 2.333 g/L under this condition;the best culture condition was 124 m L of liquid loading,10.6%inoculum,p H 4.1,and the mycelial biomass was 4.3061 g/L under this condition.2.By observing the sensory changes of the mushroom packet during the germination process,measuring the trend of reducing sugar and other physical and chemical indicators as well as the key enzyme activity in the packet,and using the mushroom yield and quality as the judgment basis,a scientific multidimensional judgment parameter and index of the maturity of ashwagandha was established.The results showed that the color of Gr0001+3 bag changed from creamy white to yellowish brown at50 d-70 d,and the texture from the top to the bottom was gradually softened;during the cultivation process,the p H,reducing sugar and soluble protein contents of the upper,middle and lower part of the bag differed greatly at the early stage of maturation,but tended to be the same at the late stage of maturation,and the xylanase activity gradually increased from 9 U to 21 U.Manganese peroxidase activity increased and then decreased,carboxymethyl cellulase increased sharply at 55 days,laccase activity showed a decreasing trend at the post-ripening stage and then increased again at the substrate formation stage,amylase and neutral protease activity showed an overall downward trend,and the maturation of the bag was the process of unification of the enzymatic environment within the bag;35-55 d bag opening had no effect on substrate yield,60-70 d bag opening had a significant effect on the time of substrate formation.The biological efficiency of mushroom emergence was the highest at 60 d bag opening.3.By crossing white G.frondosa Gr0001+3 with high-yielding G.frondosa Qing ash 182 in a single spore cross,the mushrooms were screened several times and examined at the mushroom and molecular levels.The average yield of the hybrid strain L5-3 was as high as 281.91g/bag,and the bioconversion rate was as high as 56.38%,and the yield was 89%and 27%higher than that of the parents Gr0001+3 and Qingshui182,respectively;the color of the hybrid strain L8-1 fruiting body was highly similar to that of the white parent,and the average yield was as high as 264.91 g/bag,and the bioconversion rate was 52.98%.The average yield was as high as 264.91 g/bag and the bioconversion rate was52.98%.The crossbred strain L10-3 had excellent fruiting characteristics,the color was like magnolia,beautiful opening and the highest yield of single flower was more than 300 g.All the three crossbred strains showed significant superparental advantage.3.The differences in gene expression in four consecutive growth stages of G.frondosa substrates under factory cultivation conditions were investigated by Illumina sequencing technology and their biological functions were analyzed.The results showed that the transcriptome profiles of the first three growth periods were more similar than those of the fully mature cotyledon stage.We found that the growth and development of G.frondosa substrates mainly involved carbon and nitrogen metabolism,amino acid metabolism,and biosynthesis.In addition,environmental factors such as CO₂ and temperature may play a key role in the induction of progenitor formation and sexual reproduction stages.In summary,this study optimized the culture conditions of the liquid strains of G.frondosa,established scientific and reliable intrinsic physiological parameters and biochemical indicators for the maturity of G.frondosa bales,explored the molecular mechanism of the growth and development of G.frondosa cotyledons through developmental transcriptomics,and selected three light-colored high-yielding strains with factory production potential,which provide reference for the factory cultivation technology of G.frondosa and lay the foundation for the innovation of G.frondosa germplasm resources.The foundation for the innovation of ashwagandha germplasm resources was laid.