Abstract <p>This study presents the development of an efficient cell suspension culture system using calli derived from coconut embryonic shoot meristems to address the bottleneck of tissue culture recalcitrance in coconut. The primary aim was to optimize inoculum selection and identify key factors influencing embryogenic potential and proliferation rates in coconut suspension cultures. Five types of calli and three inoculation techniques were compared to establish a uniform and viable cell suspension system. The findings revealed that embryogenic calli containing initial somatic embryos produced the most robust cultures. Among the inoculation methods, direct inoculation proved to be the most effective, reducing browning and contamination. Regular subculturing onto fresh medium every two days significantly minimized oxidative browning by lowering phenolic compound accumulation, thereby enhancing embryonic cell proliferation. Eighteen media formulations were systematically evaluated, focusing on combinations of 2,4-D, picloram, and meta-topolin. Among these, media supplemented with picloram with meta-topolin and 2,4-D with meta-topolin demonstrated the highest cell proliferation rates and embryogenic differentiation. In addition, cells in medium supplemented with picloram with meta-topolin exhibited lower oxidative stress, reduced phenolic exudation, and more stable physicochemical properties over time. Statistical analyses using repeated measures ANOVA confirmed that treatment type and culture duration significantly influenced cell growth dynamics. This optimized suspension culture protocol provides a reliable foundation for coconut tissue culture, facilitating future genetic and metabolic engineering advancements for this economically significant crop.</p>