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Ganoderma multipileum is a medicinal mushroom possessing potent medicinal secondary metabolites. However, in order to improve metabolic activity via molecular breeding, optimized protoplasting, regeneration and genetic transformation systems are mandatory but remain unexplored in G. multipileum. Thus, we sought to fill this gap by testing different parameters for protoplasting, regeneration, and genetic transformation based on the carboxin resistance marker gene. According to our results, the best viable protoplasting conditions were pH 6.0, mycelia age 4 days, enzyme concentration 7.5 mg/mL, 0.6 M sucrose osmoticum and 3 h of enzyme digestion. In a transformation study, a carboxin resistance gene cassette was constructed using PCR mutagenesis and restriction digestion of the native succinate dehydrogenase subunit B gene in G. multipileum. A PCR-restriction fragment length polymorphism strategy was also applied by PCR point mutagenesis to create an AgeI restriction site. The transformation system using carboxin resistant cassette proved amenable. The Sdh gene structure showed an intron structure similar to that of other Basidiomycetes and Ascomycetes. Alignment and analysis of the Sdh gene sequences in numerous fungi revealed conservation at a carboxin resistant point mutation site that could be applied to a wide range of fungal species. Further Sdh gene phylogenetic analysis revealed congruence with species phylogeny but with low bootstrap supporting values.

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