Droplet-based microfluidics platform for investigation of protoplast development of three exemplary plant species

Microfluidic technologies offer powerful tools for miniaturized and highly controlled biological experiments, yet their application in plant research remains underexploited. In this study, we present a droplet-based microfluidic platform tailored for the encapsulation and cultivation of plant protoplasts, enabling long-term observation of cell development at nearly single-cell resolution. Protoplasts isolated from leaves of Nicotiana tabacum, Brassica juncea, and Kalanchoe daigremontiana were used to evaluate the platform's suitability across diverse plant species. Our results demonstrate species-dependent responses to microfluidic cultivation, with tobacco protoplasts showing the highest viability. The system permits dynamic tracking of cell fate within individual droplets and supports the quantification of stochastic and concentration-dependent responses to chemical stimuli. Using tobacco protoplasts, we further investigated the effect of low concentrations of cytokinins (BAP) and auxins (NAA) for the early protoplast culture, up to the first division. Low concentrations (20-80 µg·L⁻¹) significantly enhanced cell survival and cell growth, while higher doses did not yield additional benefits. This work underscores the potential of droplet-based microfluidics as a high-resolution, low-volume platform for protoplast-based assays and dose-response screening, with applications across diverse plant biotechnology studies.