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Figure 1. 

Construction of a NaN₃-induced mutant library of Ganoderma lucidum and screening for triterpene overproducers.

Figure 2. 

Determining the LC₅₀ through a NaN₃ concentration gradient in Ganoderma lucidum protoplasts. (a) Mutagenesis and regeneration culture of protoplasts at different NaN₃ concentration gradients (0, 1, 2, 3, 4, and 5 mM). (b) Survival rate (%) of G. lucidum at different NaN₃ concentrations. The error bars indicate the standard deviations from three independent samples. ** p ≤ 0.01 indicates statistical significance, *** p ≤ 0.001 indicates statistical significance compared to the control group (CK). CK: G. lucidum strain 'Xianzhi No. 2'. (c) Scale-up cultivation of single colonies at the semi-lethal NaN₃ concentration.

Figure 3. 

Screening, cultivation, and selection of high-triterpenoid-yielding strains from NaN₃-mutagenized Ganoderma lucidum. (a) Screening of G. lucidum mycelium with antagonistic phenotype. The grey circles represent the wild-type 'Xianzhi No. 2' strain; the red circles represent the wild-type 'Xianzhi No. 3' strain; and the numbers represent NaN₃-induced mutant strains. (b) Screening and statistical analysis of antagonistic phenotypes in sodium azide-mutagenized Ganoderma strains. (c)−(e) Mycelial, photographic documentation, and observation of G. lucidum primordia and fruiting body growth stages.

Figure 4. 

Statistical analysis of total triterpenoid content in mutagenized strains. (a) Volcano plot of total triterpenoid content between G. lucidum high-triterpenoid-yielding mutants and the control group (CK). CK: G. lucidum strain 'Xianzhi No. 2'. This volcano plot illustrates the differential expression profiles of total triterpenoid content across 275 mutagenized strains. The x-axis represents the log₂fold change |log₂FC|, reflecting the relative abundance changes of triterpenoids between the two groups; the y-axis represents the −log₁₀ p-value, which is used to evaluate statistical significance. Different metabolites with distinct significance levels and fold change magnitudes are distinguished by markers of varying colors and shapes in the plot: Red dots: represent strains with significant upregulation (p < 0.05 and log₂FC > 1); Gray dots: represent strains with no substantial fold change or non-significant differences (|log₂FC| ≤ 1 or p ≥ 0.05); Green dots: represent strains with significant downregulation (p < 0.05 and log₂FC < −1). (b) Strains with significant differences in total triterpenoid content. Data are presented as mean ± standard deviation (mean ± SD) with three biological replicates per group (n = 3). The independent-samples t-test was performed to analyze the significance of differences between mutant strains and the control group (CK). * p ≤ 0.05 indicates a significant difference, ** p ≤ 0.01 indicates a highly significant difference, *** p ≤ 0.001 indicates an extremely significant difference, and ns indicates no significant difference (p > 0.05).

Figure 5. 

Mutagenesis breeding with sodium azide and antagonistic screening of G. lucidum strains with high total triterpenoid content.