Transient uidA gene expression in electroporated cotyledonary protoplasts of Pinus nigra ssp. salzmannii and in bombarded cotyledons

Abstract

We have developed a method for the routine isolation of Pinus nigra Arn. ssp. salzmannii (Dunal) Franco protoplasts. The optimized electroporation conditions for uidA gene (gene for beta-glucuronidase) expression in protoplasts from cotyledons excised from 8-day-old seedlings were determined using an exponential pulse wave generator for gene transfer. The protoplasts were electroporated with plasmids containing the chimeric uidA gene under the control of several promoters, and in parallel, cotyledons were bombarded with the same constructs using a biolistic gun. Both techniques confirmed that gene expression was higher when controlled by the sunflower polyubiquitin gene promoter than by the cauliflower mosaic virus CaMV35S promoter, whereas the rice actin and maize alcohol dehydrogenase promoters resulted in lower uidA expression levels, as determined fluorometrically. In this study, the electroporation procedure has been more effective than the particle bombardment procedure to determine the promoter strength on transient gene expression in P. nigra.