OPTIMIZING SURFACE STERILIZATION METHOD FOR INITIATION OF BITTER CASSAVA CALLUS CULTURE

Abstract

Surface sterilization is essential in explant preparation for in vitro cultivation to reduce contamination risk. This study focused on optimizing surface sterilizing technique to initiate bitter cassava callus culture (Rayong cultivar) for starch production based on the effects of plant growth regulator (PGR). Bitter cassava is widely used in the food and non-food industries thus, its demand is forecasted to be high in the next several years. Farmers faced many challenges in large-scale cassava plantations such as lack of infrastructure, cassava diseases and poor climatic conditions. Plant tissue culture is proposed due to its advantages such as high yield in a shorter time compared to the traditional method and the cultivation can be done outside the plant’s season. Soaking time in 70% ethanol (min), concentration of sodium hypochlorite (%) and soaking time in sodium hypochlorite (min) were selected for optimization of surface sterilizing condition of cassava explants (leaf and stem). Face Centered Central Composite Design (FCCCD) under Response Surface Methodology (RSM) in Design-Expert v9.0.6. was used for designing experiments and optimization purpose to minimize the percentage of contamination. Next, different concentrations of 2,4–dichlorophenoxyacetic acid (2,4-D) (5, 10, 15 and 20 mg/L) were supplemented to Murashige and Skoog (MS) medium to investigate their effect on callus initiation.  Based on the results, soaking cassava explants for 5 minutes in 70% ethanol followed by 10 minutes in 5.75% in sodium hypochlorite gave the least percentage of contamination (16.67% for the leaf and 25% for the stem). The highest frequency of callus formation (41.67%) was achieved when using 5 mg/L of 2, 4-D from stem explant. The results from this study can serve as a starting point in establishing bitter cassava callus culture for starch production.