Enhancement of short- and medium-term expression of transgenes in embryogenic suspensions ofPicea abies(L.) Karst

Abstract

Biolistic transfer and subsequent reporter gene expression was enhanced 3-20-fold in embryogenic suspension cultures of Picea abies by treatment with osmotica before and after bombardment. Myoinositol was more effective than mannitol + sorbitol and about as effective as sucrose in enhancing expression 1 d after bombardment. For both myoinositol and sucrose the optimal concentration was 0,25 M. Abscisic acid interacted with myoinositol to give a further enhancement. Maintaining the cells on 0.25 M myoinositol after bombardment enhanced the expression measured 10 d later. These results provide evidence for a metabolic component of the mechanism of osmotic enhancement in addition to an effect depending on partial plasmolysis at the time of bombardment. Expression driven by the polyubiquitin promoter 10 d after bombardment was more than twice that driven by an enhanced 35S, and 20 times that driven by a simple 35S promoter. Experiments using plasmids with and without scaffold attachment regions indicated that a greater proportion of the cells that expressed the reporter gene at 6 d compared with 1 d after bombardment had been integratively transformed