Protoplast from <Emphasis Type="Italic">β</Emphasis>-carotene-producing fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>: Preparation,regeneration and validation |
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Authors: | Ye Li Qipeng Yuan Xueling Du |
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Affiliation: | (1) Key Laboratory of Bioprocess of Beijing, Beijing University of Chemical Technoloy, Beijing, 100029, China |
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Abstract: | The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature,
pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic
stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as
7.48×106 protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0–6.0 with digesting for 14–16 h at 28 °C. After
purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method.
To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation
for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable
in further gene manipulation experiments.
This work was presented at 13
th
YABEC symposium held at Seoul, Korea, October 20–22, 2007. |
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Keywords: | Blakeslea trispora Protoplast Preparation Regeneration Validation |
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