One-to-one Fusion of Plant Protoplasts by Using Electrofusion Based on Electric Field Constriction

In plant breeding, efforts have been made to efficiently obtain plants with desired characteristics through methods such as mutagenesis, gene recombination, genome editing, and cell fusion. Among these methods, cell fusion has been widely used because of its simplicity. However, traditional fusion methods suffer from a lack of controllability and selectivity with regard to the fusion of target single cells. In this study, a microfluidic platform that enables electrofusion based on electric field constriction was developed to achieve one-to-one fusion of plant protoplasts derived from flowers (Phalaenopsis) and leaves (Phalaenopsis and Raphanus raphanistrum subsp. sativus). The platform has a simple configuration, which comprises an insulating orifice sheet sandwiched between two indium tin oxide glass electrodes, which are separated from each other with a thin polydimethylsiloxane (PDMS) separating layer. Prior to fusion, protoplasts are introduced into the chambers above and below the orifice sheet, followed by the application of a high-frequency electric field (1 MHz, 1 × 10⁴ V/m) to attract the protoplasts into the orifice by positive dielectrophoresis (pDEP). Upon confirming the formation of protoplast pairs at the orifices, a pulse voltage (5 kHz, 4 Vpp with a duration of 200 μs) is then applied to electrically fuse the paired protoplasts one-to-one at the orifice. As a result, we observed Phalaenopsis and R. raphanistrum subsp. sativus protoplast pairing with high-efficiency levels of 75% and 78%, respectively. One-to-one fusion efficiency of the paired protoplast was 76% and 80%, respectively. These results suggest that our platform has the potential to achieve high-efficiency one-to-one fusion, regardless of the protoplast type and size differences.