like for N. tabacum: you peal the epidermis from the lower part of the leaf and rest the "open leaf" on enzyme mix, then follow a classic protoplast isolation procedure. Do you have those protocoles?

amicalement,

Nemo

Thanks Nemo,

But I thought that agro was transforming only epidermis. Do you recover some agro-transformed protoplast using this method? And no I don't have those protocols: can you send them to me please?

Cheers,

MCC

Sorry, i forgot to mention, that I have done this after agro infection, and you'll find transformed protoplasts (from mesophyll cells) no problem as agro infects ALL available cells, not just the epidermis. I don't have your email for sending protocols, but you might have mine?

Cheers

Nemo

@ Nemo, why do we need to peel the epidermis from the lower part of the leaf ? When we did it, we just cut up the leaves and place them into the Protoplasting enzyme solution overnight. 

@Marie, does this experiment intend for 'transient' assay? Or you intend to culture the transformed protoplasts into transgenic plants? I guess you just want a transient assay.

Hi Marie,

You can actually try to peal your leaf, I also did that on hole plant and then you looking for fluorescent protoplast later. I have a protocol to do Benth protoplast and I send you this one. I also tried protoplast transfection wit PEG method, unfortunatly without any success.

Cheers