Hello everybody,
I am doing a research related to protoplast isolation in one species of filamentous brown algae. Now, I m testing several parameters in order to obtain high amount protoplast. The problem is that I m only using one strain for all my experiments and, in consecuence, all my replicates are technical ones. So, I was planning, first, to optimize the process only in one strain (of course, all the conclusions will be reduced to this unique strain) and then test my optimized protocol in several strains from the same species (in this case, I will have true biological replicates). Is this design sounds good for you? Or do i have to optimize the isolation using all the strains since the beginning?
Note: my strains are from different localities, so this factor could affect also the amount of protoplast that I can obtain!
Many thanks!
I never worked with brown algae but I developed protocols for protoplast isolation for several plant species. You should rapidly compare several strains with the best protocol that you have identified until now, even if it is not optimal. The idea is to verify that the strain that you use for the optimization is representative of the other strains. You should work with a strain with an average behavior (not the easiest, not the worst).
Thanks CRK Reddy :)! Do u mean the cell wall composition of each strain?
Yes. The selection enzymes for preparation protoplasts directly depends on the type of cell wall composition of the strain. The enzymes you chose for the preparation of protoplasts should have ideally all those capable of dissolving all structural cell wall polysaccharides. Otherwise, you may end with cells with rudiments of cell wall polysaccharides, if succeeded isolation.
Thanks so much :)...by the way....is bursting assay a good choice for asses (indirectly) the lack of cell wall instead of Calcofluor staining?
hi there,
your overall aspect of standardizing a common protocol for protoplast isolation is good, but in my point if your samples are from distinct places at first biological parameters (like pH, salinity, temp) and cell wall components assessment like CRK sir said previously, both are necessary.Then based on the parameters, you can find out the habitual nature of those strain and later by physical or chemical stress you can isolate the protoplast using previously reported methods as references. Hopefully you might have succeeded by this time...any ways wishes and congrats for your work
Vidyadharani.G
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence