A design-of-experiments framework for protoplast culture media optimization

Plant protoplast culture is a critical enabling technology for genome editing, functional genomics, and plant regeneration, yet culture medium optimization remains largely empirical due to the large number of interacting variables. Here, we implemented a sequential Design of Experiments (DoE) workflow to systematically optimize early Brassica napus protoplast culture. Fractional factorial screening identified osmolarity, 2,4-dichlorophenoxyacetic acid (2,4-D), and basal salt composition as the principal determinants of early viability among eleven candidate factors. A constrained ion-substitution approach combined with multivariate modelling further resolved basal medium effects and identified calcium availability as the dominant ionic driver of viability, leading to the selection of an MS-based formulation supplemented with calcium chloride. These factors were refined using response surface methodology with simultaneous optimization of viability, viable concentration, and early cell division. Experimental validation confirmed the predicted response trends and demonstrated that the optimized formulation increased viability and significantly improved day-14 cell mass relative to the reference medium. Together, this study demonstrates a DoE workflow for protoplast medium optimization that reduces experimental complexity while identifying biologically meaningful culture parameters, providing a practical framework for systematic protoplast culture medium optimization.