Culture and chemical-induced fusion of tobacco mesophyll protoplasts in a microfluidic device

Microfluidic systems provide a powerful platform for biological analysis and have been applied in many disciplines. However, few efforts have been devoted to plant cell study. In this article, an optimized culture of tobacco mesophyll protoplasts and their first polyethylene glycol-induced fusion in a microfluidic device are presented. Culture medium optimization and dynamics of protoplast growth including size change, organelle motion, and cell mass formation were also investigated microscopically in real-time. On-chip protoplast culture showed that the first division percentage of tobacco mesophyll protoplasts could be improved as high as up to 85.6% in 5 days using NT1 medium, and the percentage of small cell mass formation was more than 48.0% in 10 days. Meanwhile, chemical-induced fusion of tobacco mesophyll protoplasts was realized in 3-5 min and a 28.8% fusion rate was obtained, which was similar to the conventional fusion in a macro-scale environment. These results will be helpful for the development of microfluidics-based studies on manipulation and analysis of plant cells in a miniaturized environment, including cell growth and differentiation, gene isolation, and cloning.