Laser‐mediated gene transfer in rice

Abstract

We report an effective system for introducing exogenous DNA into cells of embryonic calli of Oryza sativa L. cv. Japonica. Plant cells were pretreated in hypertonic buffer to draw some of the water from the cells and were then put into medium of less negative osmotic potential containing exogenous DNA and treated immediately with a laser microbeam (λ=355 nm) to puncture holes in the cell wall and membrane. The pretreatment of the cells generated a gradient of osmotic pressure between the inside and outside of the cells, which facilitated the uptake of material into cells through the laser perforations. Bright yellow‐green fluorescence could be detected inside cells that had been bathed in a solution containing the fluorescent molecule calcein. β‐Glucuronidase (GUS) genes were successfully introduced into rice cells as indicated by gene expression both in post‐treated cells and in plantlets derived from kanamycin‐resistant calli that had been treated by this method.