Effects of Different Hormone Treatments on Nonembryogenic and Embryogenic Callus Induction and Time-Term Enzyme Treatments on Number and Viability of isolated Protoplasts in Saffron (Crocus sativus L.)

In order to determine the best hormone treatment for nonembryogenic and embryogenic calli induction, apical meristem of young corms derived from saffron (Crocus sativus L.) were cultured in solid LS medium supplemented with different concentration of two auxin (NAA and 2,4-D) and three cytokinin (BAP, Kin and 2ip) at 22°C in darkness. Statistical analysis using Kruskal-Wallis Test showed that treatment containing 2 mg/L NAA and BAP with highest Mean Rank had the beat effect on induction of nonembryogenic callus and treatment containing 1 mg/L 2, 4-D and BAP had the best effect on induction of embryogenic callus. The results obtained from counting number and viability of protoplasts isolated from embryogenic calli with homeocytometer and Trypan Blue under the enzyme solution consisting of MS medium with 0.1 % (w/v) Pectolyase Y-23, 1 % Cellulose R-10, 1% Deriselase, 0.1 % MES (2-N-morpholino ethane sulfonic acid), and 0.3 M mannitol at pH 5.7 on a rotary shaker (200 rpm) at 25 °C in darkness for 4 time-term treatment (1.5, 3, 4 & 5 hour) indicated that after 3 h enzyme treatment, 40×105 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 viability of fragile protoplasts, Calsium-alginats beads were used for their immobilization.

Keywords:

immobilization , somatic embryogenesis , triploid , trypan Blue