World Journal of Microbiology and Biotechnology ( IF 4.0 ) Pub Date : 2021-06-11 , DOI: 10.1007/s11274-021-03084-5 Jegadeesh Raman 1 , Kab-Yeul Jang 1 , Youn-Lee Oh 1 , Minji Oh 1 , Ji-Hoon Im 1 , Hariprasath Lakshmanan 2 , Won-Sik Kong 1
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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.
Graphic abstract
中文翻译：
灵芝与灵芝原生质体融合的种间杂交
通过聚乙二醇(PEG)诱导融合技术，尝试了灵芝与G. applanatum之间的种间杂交。原生质体分离过程被简化，我们从两种灵芝物种中获得了大量的原生质体。得到的原生质体数为5.27±0.31×10 7 / mL，在G.灵芝和5.57±0.49×10 6 / mL，在G.舌。pH 5.8 和酶解时间 3.5 小时的渗透稳定剂 NaCl (0.4 M) 支持高频原生质体再生。G. lingzhi和G. applanatum再生频率分别为 1.73 ± 0.04% 和 0.23 ± 0.02%。40% 的 PEG 诱导大量原生质体融合，在基本培养基上再生频率为 0.09%。从以下四个单独的实验中分离出 252 个融合体集落。其中，10个融合子与其亲本和其他融合子分离株相比表现出菌丝形态差异。子实体可以在橡木锯末和麦麸基质上产生，其中一些表现出亲本菌株的重组形态。GF248 的产量和生物功效 (BE) 最高，而 GF244 最低。融合体的杂交是基于菌丝体、果实形态和 PCR 指纹图谱确定的。十个融合子和亲本的 ISSR 和 RAPD 谱分析表明融合子包含多态带，这指定了融合子中 DNA 的重排和缺失。基于 RAPD 配置文件构建了一个树状图，聚类数据展示了两个主要的聚类：聚类 I 包括G. lingzhi和 Cluster II，包括G. applanatum和 fusant 系。将总多糖（α、β 和总葡聚糖）含量与融合子和亲本菌株进行比较。本研究强调了从灵芝物种中分离原生质体的有效方法。PEG 诱导的融合子表现出较高的多态性频率指数，而表型特征与G. applanatum表现出较高的相似性。在融合子和亲本菌株之间观察到蘑菇产量及其总多糖的显着差异。
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