protoplast fusion - (Jan/12/2007 )

Hello all,

I am trying to fuse fungal protoplasts of two different strains. As fusion solution, I used 60% w/v polyethylene glycol (PEG) 8000 mixed with 3x PEG amendaments (1.8M KCl, 150mMCaCl2 and 150mMTris) and it didn’t work.

Do you have some suggestions? Thank you.

Are you spheroplasting the cells prior to PEG treatment?

yes I do, and I check the recovery (viability) of the protoplasts from each strain on separate plates. The protoplast recovery was good on both strains. Only on the protoplast fusion plates nothing grew.

When you say you are checking the viability, does this mean that you are treating them with PEG in a mock fusion reaction? What is the selection for your final fusion reaction?