Hi, Mojtaba, which plants have you used? The protocol is quite simple: put cells in the enzyme solution (0.2- 0,4 M Glucose + enzyme (pectinase) + 10mM Ca, pH 5.4) and incubate for 2-4 hours, dependent form enzyme concentration and cell types.

What was the aim of your experiments? please, consider that protoplasts may alter secondary metabolite production.

Best wishes!,

Hi, Mr. Pasternak,

I study on production of secondary metabolites  in rosemary (rosmarinus officinalis)  for my m.Sc. thesis.I just want to isolate cells from callus for suspension cell culture by enzymatic methods because mechanical methods for cell digestion cause to cell browning.My aim is to increase secondary metabolites in response to inducers.

Thanks for your advise.

Hi ,

 You have a compact callus/ a friable one? if compact, alter the hormone combinations and concentrations to obtain friable callus. friable callus can be directly used for inducing cell suspension cultures. No need to isolate single cells.

Hi, Mojtaba, just put callus for liquid medium contain 2,4-D and BAP and culltured on the shaker in the dark.

You do not need to digest cell wall.

Best wishes!

dear Pasternak,

I put my callus in MS containing 0.5 mg/L 2, 4-D in light condition but the cells didn't digest and my callus to be browned thus I want to disolve this problem by an eanzymatic method.

Hi, Mojtaba, I am not sure digestion will help.

May be you have to use more adapted medium with normal NPK ratio, dark and some cytokinin.

Dear Nair,

Thanks for you answer.I don't have time to change PGRs treatments and I want to use caslli which is available now.