Expression of luciferase and β-glucuronidase in Pinusradiata suspension cells using electroporation and particle bombardment

Abstract

The reporter gene β-glucuronidase, under the control of the 35S promoter from cauliflower mosaic virus, was introduced into a suspension-cultured cell line of Pinusradiata using electroporation and particle bombardment. Electroporation of suspension-derived protoplasts in the presence of pBI221 resulted in transient β-glucuronidase activity. Maximum levels of β-glucuronidase activity were obtained 24 h after electroporation using field strengths of 1000 V/cm but decreased to background levels within 48 h. This decrease corresponded to a drop in protoplast viability and metabolic activity, as determined by fluorescein diacetate staining and chlorophyll fluorescence. Electroporation of P. radiata protoplasts with a plasmid containing a firefly luciferase reporter gene driven by a 35S promoter resulted in expression levels that were 2–3.5 times that of background. Biolistic transfer of a β-glucuronidase coding sequence driven by a 35S promoter resulted in histochemically detectable β-glucuronidase activity in P. radiata suspension culture cells. Extracts from P. radiata suspension culture cells containing 800 μg soluble protein inhibited the β-glucuronidase activity of Escherichiacoli extracts by 50%. Aliquots of P. radiata extracts containing less than 100 μg of total soluble protein did not exhibit any inhibitory effect. These studies establish two different methods of gene transfer into a P. radiata cell line and will contribute to our ability to examine a variety of promoter types associated with transcriptional regulation in conifers.