Optimization and characterization of Bitter Cassava Callus culture for starch production

Abstract

Cassava starch, a key product of cassava cultivation, has driven the expansion of large plantations in rural areas. This has led to several issues, including insufficient infrastructure, soil degradation, and a reliance on imported labor and mechanization. Thus, this study was in place to explore plant tissue culture technology to improve starch production efficiency. The present study aims to determine the optimum culture parameters for maximal cassava callus growth and starch content, explore the ideal NaOH concentration for the highest starch yield, and analyze the extracted starch's physical, chemical, structural, thermal, and morphological characteristics. The optimization of starch production and callus growth involved a multi-stage experimental approach. It began with surface sterilization and factor screening using Plackett Burman Design (PBD), focusing on basal media (Murashige & Skoog (MS) and Gamborg B5), carbon sources (sucrose, glucose and maltose), and plant growth regulators (PGRs) (Auxin: 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA); Cytokinin: benzylaminopurine (BAP) and kinetin). Significant factors identified were further investigated using the One-Factor-At-A-Time (OFAT) methodology. The process continued with a face-centered central composite design (FCCCD) to refine the media composition and parameters based on OFAT findings. The collected callus was dried and then immersed in varying concentrations of NaOH (0.10 w/v %, 0.25 w/v % and 0.50 w/v %) to determine the optimal levels that would lead to the highest starch yield, recovery, and purity following the completion of the starch purification process. A comprehensive analysis of starch obtained from native cassava tuber (CAS) and cassava callus culture (CAL) was performed using amylose content analysis, FTIR, TGA/DTG, and SEM studies. The present study observed the optimal conditions for cassava callus growth and its starch production being 110 g/L sucrose, 9.48 g/L Gamborg B5, and 6.15 mg/L of 2,4-D, which resulted in a callus weight of 0.331 g and a starch content of 4.640 %. Alkaline extraction with 0.25 % NaOH significantly enhanced starch yield, purity, and recovery at 1.0944 %, 22.67 %, and 35.89 %, respectively, compared to control samples without alkaline treatment. The study also highlighted differences between CAL and CAS starches, with CAL starch showing low amylose content and suitability for biodegradable materials, while CAS starch exhibited higher amylose content and purity. Both types demonstrated distinct chemical structures and thermal degradation patterns, suggesting distinct applications based on their unique properties. These results highlight the possibility of using cassava plant tissue culture as an alternative method for traditional starch production by investigating various plant tissue culture techniques, including cell suspension culture.