Mechanisms for introducing 250 kDa fluorescent molecules and Cas9/sgRNA into plant cells by plasma treatment

Plant cell walls prevent molecules with high molecular weights from reaching the cell membrane, challenging genome editing in plants. To overcome this challenge, the microplasma method, established as a gene and molecule transfection technology in animal cells, was investigated in tobacco plants. We found that plasma treatment of tobacco leaves and calluses introduced fluorescent molecules into epidermal and callus cells. Scanning electron microscopy revealed that plasma treatment decomposed the cuticula layer on the surface of tobacco leaves and that plasma treatment decomposed the extracellular matrix and caused cracks on the cell wall surface of tobacco callus. These results suggest that when external molecules are introduced into plant cells by plasma treatment, the external molecules' transport pathway reaches the cell membrane by degradation of the cuticula layer and extracellular matrix. Additionally, the introduction of molecules by plasma treatment was inhibited by an endocytosis inhibitor, indicating that plasma stimulation induces endocytosis. In summary, plasma treatment decomposes the cuticula layer and cellular interstitium, allowing molecules to reach the cell membrane, after which they are introduced into the cell via endocytosis.