Improved efficiency of uidA gene transfer in stone pine (Pinus pinea) cotyledons using a modified binary vector

This paper describes the construction of a binary vector with the uidA-int gene under the control of the promoter and first intron from the maize (Zea mays L.) ubiquitin 1 (Ubi1) gene, aiming at high expression levels of the uidA gene in Pinus pinea L. cotyledons. Transient expression was determined after microparticle bombardment of 1-day-old cotyledons, and data indicated that the new construct performed considerably better than p35SGUSint, which has the uidA-int gene under the control of a CaMV35S promoter. In addition, we have demonstrated that the marker gene uidA from plasmid pBINUbiGUSint is reproducibly transferred from Agrobacterium tumefaciens (E.F. Sm. & Town) Conn, and beta-glucuronidase activity was detected 7 days after inoculation of scraped or sonicated cotyledons. Infection with A. tumefaciens EHA105 pBINUbiGUSint significantly improved results obtained earlier with plasmid p35SGUSint. Further, the application of our protocol reduced the level of the necrotic response that is usually displayed by cotyledons after Agrobacterium infection.