Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 10⁷/mL in G. lingzhi and 5.57 ± 0.49 × 10⁶/mL in G. applanatum. Osmotic stabilizer NaCl (0.4 M) at pH 5.8 and enzymolysis time 3.5 h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, β and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.