Hi, llika. You can follow the protocol which available on professor Jen Sheen' website ( Harverd University). She poster very useful protocols and video about how to isolate protoplast from leaves on her website. Try to search "study signal Transduction in Arabidopsis and Maize Mesophyll Protoplasts", you will not miss it. Good luck!

Thank you Ym.

What kind of plant species are you working on? The detailed steps in the protocols for different plant species (i.e. tobacco, Arabidopsis, rice, other monocots.......etc) can be different.

We have also discussed a similar question on RG recently, and the author eventually found that 'vacuum' can make a big difference. See below:

"Can anyone please help me with choosing the right method of isolating cereal protoplasts from leaves?"

See: https://www.researchgate.net/post/Can_anyone_please_help_me_with_choosing_the_right_method_of_isolating_cereal_protoplasts_from_leaves#view=5781e47793553ba2ed4231e1

Hello Dr. Yau. I need to isolate protoplasts from tobacco leaves. The paper on rice that you shared is of immense help to me. Thank you

Hi Ilika,

I attach an article we published for you. The article includes paragraphs describing the detailed steps of protoplast isolation from tobacco leaves, chemicals it needs and figures. I hope it is useful for you.

This article covers all aspects i was looking for including chemicals needed and detailed method. Thank you so much.

Try Tape sandwich method from "https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-5-16"

Thank you for the link.

Hi, I am also trying to isolate protoplasts from tobacco leaves and 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 dr. Yau's paper) 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 case, they want protoplasts to recover and form shoots, but in the other they just want to observe them as they are?

Thank you very much!