A Simple and Rapid Method for Optimizing Particle Bombardment Parameters Through Transient Expression of Gus Reporter Gene in Onion Epidermal Layer Cells

Protocol to gauge the efficiency of transformation and gene transfer parameters prior to development of transgenic plants, has been developed by analyzing the transient expression of visual reporter gene encoding $-glucuronidase (GUS) in onion epidermal cells. The onion epidermal layerfs) were independently bombarded by coating tungsten particles with plasmid pWRG2426 containing GUS gene under the control of Cauliflower Mosaic Virus (CaMV 35S) promoter. The GUS gene expression was assessed within 24 h following particle bombardment by counting the number of islands of cells showing indigo coloration and the measure of intensity was based on relative indigo color development among the bombarded cells, which is representation of efficiency of transgene expression and transformation parameters. Although the optimum stable transformation conditions may be different from transient expression, because cells that score positive in transient expression upon bombardment may not devide to produce stable transformant, however transgene expression studies can be used as a tool to gauge efficiency of transformation parameters at preliminary stage of the experiment.