Effects of Different Hormonal Treatments on the Callus Production and Plantlet Regeneration in Saffron (Crocus sativus L.)

Abstract

Saffron (Crocus sativus L.) is one of valuable and native plants in the land of Iran. By this investigation the best hormonal compositions for callus production from protoplast and for plantlet regeneration from callus were determined. To isolate protoplasts, the embryogenic calli were used. The embryogenic calli were immersed in enzymatic solution to degrade the cell walls. The treated mixture was filtered and then centrifuged at 100 g for 3-5 min and the resulted pellet was rinsed. After one step of washing and another step of centrifugation, the protoplasts were gently mixed with sterile sodium alginate solution and added to MS broth consisting 1% calcium chloride and 0.3 M manitol to form calcium alginates granules. The protoplast-containing granules were exposed to MS broth including 0.3 M manitol and various treatments of two kinds of auxins (2, 4-D and NAA) and three kinds of cytokinins (2ip, Kin, BAP), respectively in four rates of 0.1, 0.2, 0.5, 1.0 mg L(-1) for auxins and in three rates of 0.1, 0.2 and 0.5 mg L(-1) for cytokinins, incubated in dark at 22 +/- 2 degrees C for a period of 30 days. Out of all the treatment of 2, 4-D (1.0 mg L(-1)) and Kin (0.2 mg L(-1)) was the best in callus induction. In order to regenerate plantlets, the resulted calli were transferred to MS broth amended with different rates of ABA (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg L(-1)) so that they could pass the steps of embryonic maturation. The mature embryos were transferred to MS media with different rates of GA3 (0.0, 5.0, 15.0, 20.0, 25.0 and 30.04 mg L(-1)) to initiate germination. The germinated embryos were then placed in solid MS media with various rates of NAA and 2, 4-D auxins (0.0, 0.5, 1.0 and 2.0 mg L(-1)) and different levels of BAP and Kin cytokinins (0.0, 0.5, 1.0 mg L(-1)). Results from statistical analyses indicated the treatment of NAA and BAP (each 1 mg L(-1)) as the best hormonal treatment for the plantlet regeneration from the domestic saffron calli.