FACTORS AFFECTING TRANSIENT GENE-EXPRESSION IN CULTURED RADIATA PINE COTYLEDONS FOLLOWING PARTICLE BOMBARDMENT

Transfer and expression of the beta-glucuronidase gene (gusA) in cultu red cotyledons of radiata pine (Pinus radiata D. Don) were obtained by particle bombardment. Conditions for optimum transient expression wer e established by using plasmid pBI121, delivered by gold particles, 1. 6 mu m in diameter, into 8-day-old cultured cotyledons. Helium pressur e of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 mu g of plasmid DNA per bombardment p roved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 b lue spots per cotyledon. This system was used for studying the express ion of several gus-driven promoters; the expression of the sunflower u biquitin gene promoter was higher (99% of positive cotyledons, with 14 .2 blue spots per cotyledon) than that of the CaMV 35S promoter, where as the rice actin and the maize alcohol dehydrogenase gene promoters g ave lower gusA expression, as determined histochemically. These result s were confirmed by using the gus fluorometric assay. Use of the sunfl ower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus-expressing loci pe r bombarded cotyledon, whereas with the CaMV 35S promoter gusA express ion was lost 12 days after bombardment.