Antrodia cinnamomea, T. T. Chang & W. N. Chou, T. T. Chang & W. N. Chou

Liu, Xiaofeng, Xia, Yongjun, Zhang, Yao, Liang, Lihong, Xiong, Zhiqiang, Wang, Guangqiang, Song, Xin & Ai, Lianzhong, 2021, Enhancement of antroquinonol production via the overexpression of 4 - hydroxybenzoate polyprenyltransferase biosynthesis-related genes in Antrodia cinnamomea, Phytochemistry (112677) 184, pp. 1-9 : 6

4.4. Protoplasting of A. cinnamomea S-29

Ten milliliters of the spore suspension (containing 1 × 10 8 spores/ mL) of A. cinnamomea S-29 were inoculated in a 500-mL flask with 100 mL of the seed medium. The suspension was incubated at 28 ◦ C and 100 rpm for 4 days. Preparation of protoplasts was carried according to the reports ( Yu et al., 2016; Wu and Chou, 2019) with some modifications. The germlings were collected by centrifugation at 3000 g for 10 min and then rinsed with osmotic stabilizer (0.8 M sucrose, pH 6.0) 3 times. A predefined workable protocol for A. cinnamomea S-29 protoplasting is 4-day-old germlings, mixed enzyme 20 mg /mL (lysing enzyme L1412, snailase, and cellulase, Sigma-Aldrich, USA), 0.8 M sucrose (pH 6) as osmotic stabilizer and shaking at 28 ◦ C, 4 h, and 80 rpm for digestion. In (2016) with some modifications. The 100 μL of ice-cold 25% PEG solution (polyethylene glycol MW 4000, 10 mM Tris buffer, 10 mM CaCl 2, pH 7.4) containing 3 μg, 5 μg, and 10 μg of ClaI linearized plasmid DNA was added to 200 μL of protoplast suspension (10 6 protoplasts/mL) in an Eppendorf tube, respectively. After mixed briefly, the sample was incubated on ice for 30 min. Then, the mixture was added 1 mL of 25% PEG solution and incubated at 25 ◦ C for 30 min. The whole mixture was inoculated in 10 mL seed medium containing 0.8 M sucrose. After 48 h of regeneration (28 ◦ C, 80 rpm), the seed culture were centrifuged at 2500 g for 10 min and spread on PDA containing 20 μg/mL of hygromycin B for selection of putative transformants.