Expression of GUS and CAT activities using electrotransformed pollen

Abstract

Two plasmid constructs, carrying either the gene encoding β-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) have been successfully incorporated into electroporated pollen from tobacco. The procedure employs the electroporation of germinating pollen in the presence of foreign DNA using an exponential discharge pulse. When the pollen tube membrane is permeabilized by the electroporation pulse, it allows the influx of DNA that has been added to the electroporation medium. Species-specific modifications of the electroporation medium maintain the viability of germinating pollen while optimizing the DNA uptake. Transient assays of CAT and GUS expression in pollen were used to monitor changes in uptake of DNA to optimize DNA expression by electroporated pollen. One DNA construct, pLAT52-7, contained a pollen-specific promoter, which enhanced GUS expression in pollen as compared with experiments using pBI221 plasmid with the CaMV 35S constitutive promoter. Production of transformed plants by pollination with electroporated pollen was confirmed by genomic DNA Southern hybridization, PCR amplification and hybridization, fluorometric GUS assays, and tissue level histological localization of GUS expression. The introduction of genetic material into the pollen and the production of transformed plants produced from seed formed after fertilization with treated pollen could have a tremendous impact on the improvement of economically important crops.