A laser microbeam as a tool to introduce genes into cells and organelles of higher plants

Abstract

AbstractA number of methods have become available to introduce genes into animal cells. In plants the rigid cell wall has presented a substantial barrier to similar efforts. Using protoplasts represents one way to overcome the obstacles to gene transfer. In many species these wall‐less cells, however, are sensitive to manipulations and do not regenerate to plants. Furthermore, direct gene transfer into organelles is even further complicated by the fact that DNA has to pass through two membrane systems. —Recently a method has been developed to deliver DNA randomly to cells with a particle gun [1]. It is very desirable, however, to introduce DNA into specifically selected cells or their organelles. —A laser beam focussed to its diffraction limits and coupled into a microscope provides a tool to cut openings of less than 1 μm into cell walls or membranes. Under visual control cells can be selected for gene transfer or for surgical manipulation [2]. Direct transformation with cloned DNA was achieved in mammalian cells [3,4], —Now this tool was used on oilseed rape (Brassica napus). With a laser microbeam DNA was introduced into cells and microspores of Brassica napus [5,6]. Individual cells were placed in the light path of the microscope and a single laser pulse was released at the cell wall and membrane. At the site of the laser focus wall and membrane were opened. At the time of the experiment the cells were incubated in a hypotonic buffer containing DNA. The nucleic acid was taken up into the cells. The opening in the membrane was only temporary and closed within less than 5 s after the laser pulse. The cells or microspores developed into colonies or embryos respectively with a frequency of 30–50% of the untreated controls. —Using the same technique DNA was also integrated into isolated chloroplasts [7]. Furthermore DNA can be incorporated into chloroplasts inside of a cell, large quantities of DNA were first brought into the cytoplasma. Then the laser beam was focussed on each chloroplast inside of the cell and a single laser pulse was released at each organelle. Again a small opening was cut into the membrane allowing the DNA to enter the organelle [6]. This opening closed again within 1.2 s as could be demonstrated by computer enhanced image analysis. —Laser microbeams provide a new powerful tool for introducing DNA into plant cells and their organelles as well as for analyzing chromosomes.