Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the beta-glucuronidase gene (uidA) in cultured c otyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bo mbardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 mu m-diameter gold particles, into 1- day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier trave l distance of 6 cm, and 0.8 mu g of plasmid DNA per bombardment, were the b est parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed beta-glucuronidase activity , with a mean of 63 Gus foci per cotyledon. This system was used to study t he expression of uidA gene driven by several heterologous promoters. The ex pression under the control of the sunflower polyubiquitin gene (UbB1) promo ter (Delta 1 deletion) was higher (99% of GUS positive cotyledons) than und er the control of the CaMV35S promoter, whereas the rice actin and the maiz e alcohol dehydrogenase gene promoters gave lower uidA expression, as deter mined histochemically. These results were confirmed by using the GUS fluoro metric assay. Use of a deletion of the sunflower polyubiquitin promoter res ulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The resul ts will be useful to design protocols for stable transformation and high le vels of transgene expression in P. pinea.