The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants.

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

An in vitro study on the postinfection activities of copper hydroxide and copper sulfate against conidia of Venturia inaequalis.

The postinfection activities of copper hydroxide [Cu(OH)2] and copper sulfate (CuSO4) against apple scab (Venturia inaequalis) were evaluated in an in vitro study. Our intention was to support the aim of reducing copper application rates by appropriate timing of applications. Experiments were conducted at 20 degrees C with leaf disks and isolated cuticular membranes (CM) of Malus x domestica 'Gloster' and 'Elstar'. Conidia of V. inaequalis were used as the inoculum. In untreated controls, 7.9 and 33.2% of germinated conidia formed primary stromata 24 and 48 h after inoculation, respectively. Treatments with copper compounds were applied 24 and 48 h after inoculation, which was 16 and 40 h after infection had occurred. When working with CM and using fluorescein diacetate as a vital stain, vital and dead stromata could be distinguished. Treatment effects were assessed 72 h after inoculation by counting vital (fluorescing) primary stromata. With leaf disks, the number of stromata was counted using KOH-aniline blue fluorescence staining. Cu(OH)2 and CuSO4 showed postinfection activity and killed primary stromata, provided that the surface of the CM was kept wet. Cu(OH)2 was more effective than CuSO4 and was able to kill all primary stromata 24 h after inoculation at concentrations of 116 and 231 mg L(-1). When Cu(OH)2 was applied at 116 mg L(-1) to leaf disks 24 h after inoculation, the number of primary stromata did not significantly differ from the control. Results indicate different modes of action for the highly water soluble CuSO4 and the slightly soluble Cu(OH)2. This supports the hypothesis that spore exudates react with insoluble copper compounds and form highly toxic copper complexes. Application of Cu(OH)2 to dry CM did not kill primary stromata. Hence, for Cu(OH)2 to exert postinfection activity, leaves must be wet. In the field, this cannot be guaranteed and a postinfection application of Cu(OH)2 cannot be recommended.