Hi, all
I am using some protocol for protoplast production from Fusarium graminearum (Fg), but I need them in a huge amount. Sometimes the protocol worked well but most of the time failed. Temperature (30c), rotation (70rpm) are almost the same for all protocol but the enzyme conditions are different. i need to know the growing condition of fusarium spore and their amount before growing on liquid medium. I would be nice if someone has any idea about hidden tips and tricks for the Fg protoplast production. Thanks in advance. The reference paper I used are following (No 3 was preferred for the experiment )
1. (PDF) A modified method for transformation of Fusarium graminearum (researchgate.net)
2. A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense | Fungal Biology and Biotechnology | Full Text (biomedcentral.com)
3. Protoplast production and transformation of morphological mutants of the Quorn® myco-protein fungus, Fusarium graminearum A3/5, using the hygromycin B resistance plasmid pAN7–1 - ScienceDirect
By enzyme treatment, but I did not experiment with Fusarium grmainearum.
Also, it differs from lab. to lab.
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sometimes it is hard to reproduce the results from other labs without some modification. I have never made protoplasts with your cell, but I have done a lot with Streptomyces strains. My experience is the enzyme treatment is the most important step because in my case it directly affect the protoplast regeneration rate. I took different time points during incubation and pick the condition that gives the best protoplasting and regeneration. Regarding scaling up, I usually do a few small scales instead of one large scale, this helps me. I am not sure if these could help you. Good luck.
Yes, exactly it varied lab to lab but the strange thing is the protoplasts are not reproducing anymore using the same condition which was previously produced a lot. I guess its a problem with enzymes.
By enzyme treatment, but I did not experiment with Fusarium grmainearum.
Also, it differs from lab. to lab.
Best reagrds
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