treatments-xml/data/A7/73/89/A7738947BE4AB84E3A4BF9B367E0FE62.xml
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4.4. Protoplasting of
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S-29
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Ten milliliters of the spore suspension (containing 1
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10
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spores/ mL) of
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S-29 were inoculated in a 500-mL flask with 100 mL of the seed medium. The suspension was incubated at 28
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C and 100 rpm for 4 days. Preparation of protoplasts was carried according to the reports (
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;
<bibRefCitation id="4B4B45A0BE4AB84D3D61F8EB6796F8CA" author="Chou, K. C. C. &amp; Wu, H. L. &amp; Lin, P. Y. &amp; Yang, S. H. &amp; Chang, T. L. &amp; Sheu, F. &amp; Chen, K. H. &amp; Chiang, B. H." box="[1048,1236,1795,1815]" pageId="5" pageNumber="6" pagination="97 - 106" refId="ref7444" refString="Chou, K. C. C., Wu, H. L., Lin, P. Y., Yang, S. H., Chang, T. L., Sheu, F., Chen, K. H., Chiang, B. H., 2019. 4 - Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea. Phytochemistry 161, 97 - 106. https: // doi. org / 10.1016 / j. phytochem. 2019.02.011." type="journal article" year="2019">Wu and Chou, 2019</bibRefCitation>
) with some modifications. The germlings were collected by centrifugation at
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3000
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for 10 min and then rinsed with osmotic stabilizer (0.8 M sucrose, pH 6.0) 3 times. A predefined workable protocol for
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S-29 protoplasting is 4-day-old germlings, mixed enzyme
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/mL (lysing enzyme L1412, snailase, and cellulase, Sigma-Aldrich,
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), 0.8 M sucrose (pH 6) as osmotic stabilizer and shaking at 28
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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
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, 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
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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
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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
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C, 80 rpm), the seed culture were centrifuged at
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for 10 min and spread on PDA containing 20 μg/mL of hygromycin B for selection of putative transformants.
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