Hi again everybody :)!
As a part of my research, I'm culturing some protoplast from brown algae (using non-axenic material) in different regeneration media and at different initial cellular densities. While i was recording the cell wall regeneration (using Calcofluor) i realized that in almost half of the protoplast that were undergoing cell wall regeneration, a cellulose "tail" started to form (like a "comet"). At the end, the protoplast were covered with the cell wall but this "tail" remained. In other cases, the protoplast didn t finish the cell wall regeneration. The percentage of this "cellulose tails" were variable in the different media and at different densities, but anyway this phenomenom appeared in all the treatments. Checking some publications i found that this could be due a loosely attachment of the cellulose to the cell membrane. However, i didn t see this phenomenom in seaweed protoplast. Has anybody seen this abnormal cell wall regeneration?
Many thanks in advance
I don't recall seeing it in tobacco protoplasts. I wonder if it relates to the different composition of the algae cell wall compared to other organisms?
Dear Teri Jo Mauch. I m not sure about that. Also, the reason could be the isolation process itself. I have seen a similar phenomenon in a paper about recalcitrant protoplast from some plant species. I ll try to submit a picture about this "cellulose tail" these days.
I am far from being a specialist in algae protoplast technology, but as far as I can remember there is a difference in the composition of cell walls in seaweeds compared to other plants (i.e. the ratio between cellulose and hemicellulose is not identical). As a result, when staining with Calcofluor White there is a parasite staining of hemicellulose as well (reported many years ago in ferns if I do not misremember).
A couple of things might eventualy help you untangle this; 1) try dual staining with Caclcofluor (more specific for cellulose) + Sulphorodamine (specific for hemicellulose), if what happened is what I suggested above you should see blue where there is cellulose while the tail should be red; 2) there's always the alternative to modify slightly your method of preparation of the Calcofluor suspension to render it closer to a "true" solution and that is increasing the concentration of the sorbitol where it is dissolved (from 0.4 to 0.5 M for example) and reducing the impact of the KOH that should be added to start disolving the Caclcofluor powder (for instance add a fewdrops of KOH 0.1 N instead of KOH 1N as usually done).
Finally, as already evoked by others, several recalcitrant species (including some woody ones and several succulent ones) have already been reported to give such a response. Unfortunately, no reason was given nor a solution for the problem proposed.
Dear Jose, I would like to know how did you overcome this problem. Thanks in advance!
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