The present study reports an efficient protocol for isolation and regeneration of protoplasts from callus of Fritillaria imperialis L. There is no published method recommended for protoplast isolation and regeneration from Fritillaria imperialis L. A range of factors, which influence the success of isolation and regeneration of F. imperialis protoplasts, were investigated. From the results obtained, callus Fresh Weight (FW) of 0.4 g produced the highest number of viable protoplasts, which was

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... 12×10 5 protoplasts g-1 FW. The highest amount of viable protoplasts (1.01×10 5 protoplasts g-1 FW) was obtained when the mannitol concentration was maintained at 9% (w/v). The best treatment for isolation of F. imperialis protoplast (1.37×10 5 protoplasts g-1 FW) was treatment with 2% cellulase and 0.1% pectinase with 9% mannitol for 8 h. For enhancement of the protoplasts division and the percentage of colony formation, different concentrations from Casein Hydrolysate (CH), 2,4-Dichlorophenoxyacetic acid (2,4-D) and Benzyl-Adenine (BA) were used. The results revealed that cell wall and colony formation were better in liquid medium than those on semi-solid medium. The highest plating efficiency (1.26×10 6 per g FW) and highest callus formation were obtained using the medium containing 0.5 mg L-1 2,4-D, 1 mg L-1 BA, and 200 mg L-1 CH. Micro-calli were formed after one month of culture. Many plantlets were formed on the calli after transfer of the proliferated calli to regeneration medium. The highest plantlet regeneration (100%) was obtained using the medium containing 0.5 mg L-1 (Naphthalene Acetic Acid) NAA and 1.5 mg L-1 BA.