HOW TO REGENERATE PLANTLETS FROM PROTOPLASTS OF FRITILLARIA IMPERIALIS

There is no special method recommended for protoplast isolation and regeneration from Fritillaria imperialis L. The present study reports the isolation and regeneration of protoplasts from callus of Fritillaria imperialis L. A range of parameters which influence the 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 1.12 × 105 protoplasts/g FW. The best treatment for isolation of Fritillaria imperialis protoplast (7.8 × 105 protoplasts/g FW) was 2% cellulase and 0.1% pectinase with 9% manitol for 8 h. For enhancement of the protoplasts division and the percentage of colony formation, different concentrations from casein hydrolysate, 2,4-D and BA were used. The results revealed that cell wall and colony formation were better in a liquid medium than those on a semi-solid medium. The highest plating efficiency (1.26×106 per gr FW) and highest callus formation was obtained by using a medium containing 0.5 mg l ?1 2,4-D,1 mg l 1 BA and 200 mg l ?1 casein hydrolysate. 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 by using a medium containing 0.5 mg l ?1NAA, 1.5 mg l ?1 BA.