Hello, I am trying to isolate protoplasts from tobacco leaves and since being new to the topic I would really appreciate your help.
I've searched for the tobacco leaves protocols and have realised that they differ in composition of the enzyme mix. Some include MS salts, Gamborg's vitamins, xyolose, sucrose, CaCl2, NAA and BA (as in Yau et al: Method for Bxb1-Mediated Site- Specific Integration In Planta) in the enzyme mix, but others don't (only MES, mannitol, KCl and CaCl2 + enzymes - e.x. Lei et al: A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability).
Is this so, because in the first cases, they want to use protoplasts for gene expressions or want them to recover into a new plant, but in the others they just want to observe them as they are?
Thank you very much!
I was having difficulty isolating tobacco leaf protoplasts.
Then I followed this protocol, which worked nicely.
https://doi.org/10.3389/fgeed.2020.627803
Following isolation and purification, all that remains is to visualize under a microscope.
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