This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Production and regeneration of G. lucidum protoplasts
Shi-lin Chen
Chen Shilin's LAB
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Production and regeneration of G. lucidum protoplasts, which is important to G.lucidum monokaryotization
Keywords
protoplast
G.lucidum have two different types hypha in mycelium--the haploid one and the diploid one. Using a protoplast regeneration method can generate an isolate from a single cell which maybe only contain a suit of chromsomes. Firstly, we digest the mycelium into a single protoplast. Then, dilute the protoplast suspension into an appropriate concentration. Third, regenerate these protoplasts on MYG medium.
Lywallzyme
Mannitol
PDB broth
MYG agar medium
Shaker
Incubator
- Inoculate mycelia into 100 ml PDB media (200 g potato, 20 g dextrose, 1 L water) in 250 ml flasks.
- Cultivate the mycelia were cultivated in the dark at 28°C under continuous shaking at 50 rpm.
- Harvest the culturesby filtering through sterile gauze.
- Place approximately 5 g of mycelia in a 50 ml tube containing 20 ml of filter-sterilized 1.5% lywallzyme (Guangdong Institute of Microbiology, China) in 0.6 M mannitol.
- Incubate the mixtureat 30°C for approximately 2 h with occasional agitation.
- Remove hyphae fragments after 2 h of digestion. MYG agar medium (2% malt extract, 0.2% yeast extract, 2% dextrose, 1.5% agar) was used for G. lucidum protoplast regeneration.
- Dilute the protoplast suspension was diluted to 1 x 104 cells/ml, and 50 µl of the diluted protoplast suspension was added to a MYG medium plate and inoculated at 25°C in the dark.
Version History
CURRENT STATUS: Posted
Version 1
Posted 11 May, 2012
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Subject Areas
Microbiology
Method Article
Production and regeneration of G. lucidum protoplasts
Shi-lin Chen
Chen Shilin's LAB
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 11 May, 2012
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Microbiology
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Production and regeneration of G. lucidum protoplasts, which is important to G.lucidum monokaryotization
G.lucidum have two different types hypha in mycelium--the haploid one and the diploid one. Using a protoplast regeneration method can generate an isolate from a single cell which maybe only contain a suit of chromsomes. Firstly, we digest the mycelium into a single protoplast. Then, dilute the protoplast suspension into an appropriate concentration. Third, regenerate these protoplasts on MYG medium.
Lywallzyme
Mannitol
PDB broth
MYG agar medium
Shaker
Incubator
- Inoculate mycelia into 100 ml PDB media (200 g potato, 20 g dextrose, 1 L water) in 250 ml flasks.
- Cultivate the mycelia were cultivated in the dark at 28°C under continuous shaking at 50 rpm.
- Harvest the culturesby filtering through sterile gauze.
- Place approximately 5 g of mycelia in a 50 ml tube containing 20 ml of filter-sterilized 1.5% lywallzyme (Guangdong Institute of Microbiology, China) in 0.6 M mannitol.
- Incubate the mixtureat 30°C for approximately 2 h with occasional agitation.
- Remove hyphae fragments after 2 h of digestion. MYG agar medium (2% malt extract, 0.2% yeast extract, 2% dextrose, 1.5% agar) was used for G. lucidum protoplast regeneration.
- Dilute the protoplast suspension was diluted to 1 x 104 cells/ml, and 50 µl of the diluted protoplast suspension was added to a MYG medium plate and inoculated at 25°C in the dark.