Conditioning promotes regeneration and transformation in apple leaf explants

The conditioning of apple shoots for several days in an appropriate liquid medium enhances the regenerative capacity of leaf explants derived from the shoots, so that adventitious buds form in high frequency. The use of conditioning enables the transformation and rapid recovery of plants from otherwise recalcitrant cultivars without the need for an extended callus phase. Conditioning has a wide range of effects on the leaf cells, including increasing the density of the cytoplasm and the complexity of vacuoles, and increasing the porosity of the cell walls from of the order of 3.5 nm to 5.5 nm. The increased porosity may aid the insertion of T-DNA through the cell wall. Initial expression of introduced genes, as judged by the histochemical assay of the β-glucuronidase gene, occurs within 2 days of inoculation with Agrobacterium, usually in groups of 2–20 cells, termed foci. The foci are most commonly composed of an intensely expressing core cell with one or more surrounding layers of less intensely expressing cells. Explants from conditioned leaves contain at least three times as many foci as the control explants. It is concluded that conditioning of apple shoots promotes the recovery of transformed plants from leaf explants by two processes: increasing the number of cells containing and expressing the introduced genes, and by increasing the probability that cells will regenerate directly to shoots. This revised version was published online in June 2006 with corrections to the Cover Date.