Optimization of the sterilization method for leaf explant Robusta BP 308 coffee in vitro

Abstract

Abstract The research aimed to know the optimal sterilizing agent for the success of the coffee callus inoculation from young leaf explant. This research was conducted at the Plant Tissue Culture Laboratory for 2 months. The explants are used from young leaves of Robusta clones BP 308. This experiment used a nonfactorial completely randomized design with three sterilization methods and was repeated 10 times. The treatments tested were as follows Method 1: Fungicide 2 gr/L, Bactericide 2 gr/L, Erythromycin 4 gr/L (20 min); 70% alcohol (3 sec); NaOCl 0.525% (10 min); NaOCl 1.05% (10 min). Method 2: 70% alcohol (3 sec), 0.1% HgCL2 (5 min). Method 3: Fungicide 2 gr/L, Bactericide 2 gr/L, Erythromycin 4 gr/L (20 min); 70% alcohol (3 sec); NaOCl 0.525% (10 min); NaOCl 1.05% (10 min); 0.1% HgCL2 (5 min). The results showed that the sterilization method used suppressed the rate of bacterial and fungal contamination, but there was no significant difference between the methods. A very significant effect was seen in the percentage of browning explants. In sterilizing agent treatment 1, there was no browning explant, while the other methods caused browning to reach 62.5%. Browning also causes differences in callus regeneration ability. The explants which were sterilized in method 1 showed a high percentage of callus (85%) and were also appear to callus faster at 11 days after inoculation.