Gametophytic expression of GUS activity controlled by the potato Lhca3.St.1 promoter in tobacco pollen

A beta-glucuronidase (GUS) gene fusion with the promoter from the potato Lh ca3.St.1 gene, encoding a light-harvesting complex protein, shows expressio n in pollen of transgenic tobacco plants. This was established by histochem ical and fluorometric assays, and confirmed by RT-PCR, Western analysis and GUS activity following gel electrophoresis, The activity is minimal in ear ly pollen development and high in mature pollen at anthesis, It shows gamet ophytic segregation in pollen grains. Pollen from tobacco plants hemizygous for a single Lhca3.St.1-GUS locus exhibited 1:1 segregation ratios, wherea s dihybrids segregated in the expected 3:1 ratio. A sequential combination of histochemical staining for GUS activity and Alexander's stain for pollen viability, offers a convenient system to eliminate artefacts in the histoc hemical staining, identify inviable pollen and accurately score pollen segr egation. The dual staining approach also confirmed gametophytic segregation in tobacco plants hemizygous for the GUS gene under transcriptional contro l of a doubled CaMV 35S promoter. Gametophytic segregation establishes that the GUS activity is determined by the genetic status of the pollen rather than of the parent plant, and indicates de novo GUS gene expression and tra nslation during pollen maturation. The unanticipated activity of the Lhca3. St.1 promoter in pollen has implications for the biosafety assessment of tr ansgenic plants intended for agricultural use. Even when a promoter is not generally considered to be active in pollen, its expression in pollen shoul d be evaluated if effects on pollinating insects or food safety concerns fo r products such as honey can be anticipated.