Enzyme-Assisted Photoinjection of Megadalton Molecules into Intact Plant Cells Using Femtosecond Laser Amplifier

Femtosecond laser photoporation has become a popular method to deliver various kinds of molecules such as genes, proteins, and fluorescent dyes into single mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery, especially for larger molecules than the mesh size of the cell wall. This work is the first demonstration of the efficient photoinjection of megadalton molecules into a cytoplasm of an intact single plant cell by employing a femtosecond laser amplifier under moderate enzyme treatment conditions. The intense femtosecond laser pulse effectively formed a pore on the cell wall and membrane of Tobacco BY-2, and 2 MDa dextran molecules were introduced through the pore. Along with the pore formation, induced mechanical tensile stresses on BY-2 cells were considered to increase permeability of the cell membrane and enhance the uptake of large molecules. Moreover, the moderate enzyme treatment partially degraded the cell wall thereby facilitating the increase of the molecular introduction efficiency.