Mechanism of macromolecular introduce into plant cells by plasma treatment.*

Gene transfection is one of the methods of plant breeding. Since a plant cell is covered with a cell wall, introducing genes directly into it is difficult. We have been developing the gene introduction method for plant cells with discharge plasma treatment. In this study, macromolecules such as plasmids were introduced into plant cells with multi-time plasma treatments.

The callus was placed statically in a 3.5 cm dish on a GND electrode. A needle electrode was placed above the callus, and plasma was irradiated directly to the cells by applying a sinusoidal voltage. Twenty-four hours after plasma treatment, the callus was stained with X-Gluc to observe the expression of GUS. GUS expression was observed on the cell treated two times, though no GUS expression was observed for single-time treatment. After the plasma treatment, the cell surface of the callus was observed by FE-SEM. The callus treated two times scraped the cell wall and other parts of the introduction barrier more deeply than the callus treated only one time.

These results suggest that by multi-time plasma treatment, the target macromolecules, such as GUS plasmid, reach the cell membrane through the cracks in the cell wall, then they are introduced into cells by endocytosis mediated by the complex stimuli of plasma.