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Stable transformation of Arabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality of cis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-old Arabidopsis seedlings were vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying various upstream regulatory regions controlling uidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3-5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV) 35S promoter, the upstream regulatory region of ribosomal protein gene L23A-1, and a temperature-inducible regulatory region (HSP101B) also from Arabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV 35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with the HSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d following Agrobacterium infiltration than those collected 3-4 d postinfiltration. [ABSTRACT FROM AUTHOR]