Investigation of the Best Time of Enzyme Treatment in Order to Isolate the Protoplast from Embryogenic Callus of Saffron (Crocus sativus L.)

Crocus sativus L. is a triploid and sterile crop that propagates vegetatively by corm. Therefore, its improvement via traditional breeding method is difficult. Protoplast culture is one of the suitable tools for breeding of this plant. Protoplasts with a high viability are potentially interesting materials, especially for somatic hybridization, transformation and selection studies. In this research, In order to extract and isolate the protoplast, embryogenic calli were suspended in enzyme solution consisting of MS medium with 0.1% Pectolyase Y-23, % Cellulose R-10, % Deriselase, 0.1% MES (2-N-morpholino ethane sulfonic acid) and 0.3 M mannitol at pH 5.7. The mixture was placed on a rotary shaker (200 rpm) at 25°C in darkness under 4 time-term treatment (1.5, , and 5 h). Counting number and viability of protoplasts isolated from embryogenic calli with homeocytometer and Trypan Blue after 1.5, , and 5 h of enzyme treatment indicated that after 3 h enzyme treatment, 0x105 protoplasts per 1 mL of suspension with 98% viability obtained that were the best time-term treatment. Finally, in order to protect and maintain the fragile protoplast, Calcium-alginats beads were used for their immobilization.