Abstract:Protoplasts were isolated from mesophyll cells of shoot cultures of gentian (Gentiana scabra Bunge). First division occurs after 4–5 days in culturing and small calli are visible to the naked eye within one month. Low temperature pretreatment of source materials is the important factor in connection with the division of mesophyll protoplasts of gentian. It seems that sustain division is within the limits of the protoplasts of small size only. Calli derived from protoplasts are being differentiated.