I'm isolating maize protoplast from etiolated leaf and transforming with the combination of plasmids to check the TF activation, for instance, construct with TF, construct with reporter. Most of the cases, it contains Luc and GFP (fusing in both the N-and C-terminal). I'm using total plasmid concentration ranging from 5 ug to 55 ug and amount of protoplast is 1/2X10^5 cells.
I'm observing the busrting of protoplast occasionally.
> How to avoid the brusting of protoplast?
> Anyone was experiencing the same issue and tricks to overcome the problem?
Since protoplasts have no cell walls, you need to handle it with extremely care. When you move the plates or swirl the protoplasts with your plasmids, you need to be very gentle. Your plasmid solution should be clean.
The problem I'm having is not an issue of handling. Because, I tried protoplast with or without plamids and electrporation, those combinations don't cause the bursting of protoplasts. The occasional bursting of protoplasts is happening only with the plasmids after electroporation.
If you have same kind of issues and solved it, please mention how you resolved that.
You may be need to use an Osmotic stabilizer with optimum concentration, which need to be fixed by error and trial. I think the cellular component concentration become increase after transformation, which might not be supported your protoplast that osmotic stabilizer you have used during isolation.
Hi Arif,
as Ujjjal said, it might be that the osmotic adjustment you use is sub-optimal for your poration conditions. If the turgor is too high,sudden damage of the cell membrane might cause rupture. Also, poration conditions might be sub-optimal damaging the membrane too much. Although, in my hands that never lead to bursting, but rather the protoplasts just died...
Ujjal Kumar Nath, would you specify about osmotic stabilizer? The current protocol I'm using, we aren't having any osmotic stabilizer apart from regular buffer system.
Klaus Eimert, that makes more sense. In some cases, we see rough PM. If you are currently using any protocol with osmotic stabilizer, please share with me.
I have attached one sample picture of prtoplast I'm having after transformation.
Ujjal Kumar Nath and Klaus Eimert
Ujjal Kumar Nath and Klaus Eimert
I've solved the problem. I got very nice transformation and could detect the GFP signal and Renilla as well. Only issue remains with Luciferase, which is most probably happening due to some issue regarding the vector. Please check the attached pictures.
But, it would be great help for future, if you kindly share your protocol.
Hi Arif Ashraf
Can you please share how you solved the problem of bursting protoplasts after transformation? I get a lot of broken protoplasts after I do the PEG mediated transformation.
Thanks!
Mainak
Hi Mainak Das Gupta
There were couple things I did to reduce the bursting of protoplasts. (1) diluted the protoplast; (2) after adding the plasmids, wait atleast 10 minutes before electroporation; and (3) tried to reduce the amount of plasmids, both the volume and concentration. It helped a lot to get good result.
Best of luck!
This is the protocol I was following.
https://bio-protocol.org/bio101/e3346
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