PEG-mediated protoplast transformation with naked DNA.

1. Introduction Direct introduction of DNA into plant protoplasts facilitates a rapid analysis of transient gene expression, as well as the generation of stably transformed transgenic plants. Transient gene expression assays performed after DNA transformation permit a comparative analysis of cisacting regulatory sequences and their function in transcriptional control of plant genes by signaling pathways mediating cellular responses to different environmental and hormonal stimuli (I). There are a number of methods for introducing DNA into plant protoplasts, but the most commonly used technique is the polyethylene glycol (PEGjmediated DNA uptake. The PEG-mediated transformation is simple and efficient, allowing a simultaneous processing of many samples, and yields a transformed cell population with high survival and division rates (2). The method utilizes inexpensive supplies and equipments, and helps to overcome a hurdle of host range limitations of Agrobacterium-mediated transformation. The PEG-mediated DNA transfer can be readily adapted to a wide range of plant species and tissue sources. In Arabidopsis thaliana, several methods of direct gene transfer to leaf mesophyll(3-5) and root-derived protoplasts (6) have been reported. They are all derived from a PEG-mediated direct gene transfer technique established originally for tobacco protoplasts by Negrutiu et al. (7). This chapter describes a method for PEG-mediated transformation of protoplasts derived from leaves, roots, and cell suspensions of A. thaliana. Leaf mesophyll protoplasts are able to regenerate after embedding into alginate, but their yield is relatively low. In comparison, cell suspensions provide an unlimited source of rapidly dividing protoplasts that can be obtained within 2-3 h and show a transient expression