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Eucalyptus polybractea (blue mallee) is the most widely harvested species in Australia for the production of pharmaceutical grade eucalyptus oil. Its use for both oil and biomass production is set to expand because blue mallee grows vigorously on semi-arid land where agricultural crop production is unsustainable. One of the most important tools for improvement of blue mallee, and for dissection of its unique traits, is an efficient method of indirect organogenesis. Our aim was to develop such a method, which is efficient for all genotypes and applicable to adult-derived explants. We also aimed to test whether genetically modified plants could be produced using this protocol. In vitro cultures were developed from three field-grown adult clones and it was found that over 95% of the resultant leaf explants initiated callus in a medium supplemented with thidiazuron and 1-naphthaleneacetic acid. The effect of the cytokinins, 6-benzylaminopurine (BA) and N-6-(2-isopentenyl) adenine (2iP) on plant regeneration from the initiated callus was compared. Ninety percent of callus from all three clones regenerated shoots in a medium supplemented with BA, whereas only one clone regenerated shoots at high efficiency (93%) with 2iP. Roots were successfully developed on regenerated shoots with high efficiency (>60%) via exposure to indole-3-butyric acid, and hardened plants were successfully established in soil. Leaf explants from one clone were transformed with Agrobacterium tumefaciens T-DNA containing genes for hygromycin phosphotransferase (Hpt) resistance and green fluorescent protein (GFP), both constitutively expressed using the CaMV35S promoter. Transformants showed GFP fluorescence in calli and plantlet leaves and were successfully rooted and hardened into plants. This work will enable large scale cloning of adult blue mallee plants and, after optimization of the transformation protocol, provide an important platform for further research on the biosynthesis of eucalyptus oils with the potential to dramatically improve blue mallee as a crop. (C) 2016 Elsevier B.V. All rights reserved.