T A Protocol for the Efficient Screening of Putatively Transformed Plants for bar, the Selectable Marker Gene, Using the Polymerase Chain Reaction Joan E. Vickers/ Glenn C. Graham, and Robert J. Henry E-mad: Joan.Vickers@cnetns.tcp.csiro.au (JEV) Department of Biochemistry, University of Queensland, St. Lucia, QLD 4072; (GCG) CRC for Tropical Pest Management, Gehrmann Laboratories, University of Queensland, St. Lucia, QLD 4072; (RJH) Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Austraha Key words: bar gene, barley, PCR screening, transformation Abstract: Amplification of the bar gene using Taq DNA polymerase in PCR is often not successful, possibly due to bar's high GC content. We describe a PCR protocol in which reliable amplification at a sensitivity of one gene copy per genome (in this study, barley) present in the reaction was achieved using a novel pair of primers and Expand TM High Fidelity DNA polymerase mix (Boehringer Mannhelm). This method should allow for rapid screening of plants putatively transformed with bar. T he bar gene of Streptomyces hygroscopicus, which encodes an en- zyme (PAT) conferring resistance to the herbicide bialaphos, is a useful marker for selection of transgenic plants (Thompson et al., 1987; White et al., 1990). A sensitive and reliable PCR-based assay for the ~Current address, and address for correspondence: CSIRO Division of Tropical Agriculture, St. Lucia, Queensland, Australia 4072 Abbreviations: bar, gene encoding resistance to the herbicide bialaphos; CTAB, hexadecyltrimethy[ammonium bromide; DIG, digoxygenin; PAT, phosphino- thricin acetyI transferase; pPATO, plasmid containing the bar gene; Taq, Thermus aquaticus. 363