Regeneration and transformation of Eucalyptus camaldulensis

Abstract

Reliable regeneration protocols for Eucalyptus camaldulensis using leaf explants from in vitro-grown plants have been developed. Out of the 24 clones tested 13 were regenerated and of these, 6 showed regeneration from more than 60% of the explants. Identical protocols were also successful in the regeneration of some clones of E. microtheca, E. ochrophloia, E. grandis and E. marginata, but at lower frequencies. Co-cultivation of E. camaldulensis leaf explants with Agrobacterium tumefaciens strains carrying a kanamycin resistance gene and the reporter gene β-glucuronidase (GUS), followed by selection on kanamycin at 9 mg l^–1, allowed the selection of transformed shoots that could be rooted on selective media. Transformation of the plants was verified by staining for the GUS enzyme in various plant tissues, NptII assays and by Southern blotting on isolated DNA using specific probes for both the GUS and selectable marker genes. Transformed tissue was obtained with 5 clones of E. camaldulensis tested and a number of A. tumefaciens strains. However, only 1 clone regenerated transformed whole plants reliably.