Optimization of Preparation and Sterilization of Culture Medium for Callus Proliferation and Plant Regeneration in Three Iranian Commercial Cultivars of Sugarcane (Saccharum officinarum L.)

Microbial contamination (fungal and bacterial) of culture medium is one of the most common problems in plants in vitro micropropagation. In the present research, achievement of a reliable sterilization method, optimization of callus proliferation and plant regeneration medium in three Iranian commercial cultivars of sugarcane, CP48-103, CP69-1062 and CP57-164 were studied. For sterilization, 12 treatments were separately examined for each cultivar in a completely randomized design with 12 replications. In order to select the best callus culture and plant regeneration media, five treatments including MS medium supplemented with a combination of 2,4-D (2 mg l-1) and different levels of BAP and fifteen treatments using MS mediumcontaining different levels of auxin and cytokinin were studied, respectively in a completely randomized design with 10 replications.  Based on the results, disinfection treatments containing carbendazim 2 parts per thousand (15 min), 70% ethanol (50 seconds), 25% sodium hypochlorite (5% active chlorine) plus Tween 20 (15 min), and culture medium containing cefotaxime (200 ppm) and gentamicin (5 ppm) showed to be the best sterilization method without any contamination in CP69-1062 and CP57-164 cultivars and the lowest percentage of contamination in CP48-103. For callus culture, MS media containing 2,4-D (2 mg l-1) + BAP (0.5 mg l-1) exhibited the highest callus growth rate and proliferation. The highest percentages of plant regeneration were observed in treatments (Kinetin = 0.5 mg l-1 and BAP = 0.5 mg l-1), (Kinetin = 3 mg l-1 and NAA = 0.5 mg l-1) and (Kinetin = 3 mg l-1 and NAA = 0.1 mg l-1) with the means of 70%, 60% and 50% in CP48-103, CP57-164 and CP69-1062 cultivars, respectively.