Agrobacterium-mediated transformation of a Eucalyptus camaldulensis x E-tereticornis hybrid using peeled nodal-stem segments with yeast HAL2 for improving salt tolerance

Genetic improvements of Eucalyptus require both breeding and gene transformation to generate desirable traits for forest industry. Because most of the current Eucalyptus transformation methods rely on juvenile seedling tissues as explants, the resulting transgenics are subjected to phenotypic segregation through the use of segregated seedlings. This is unsuitable for transformation of selected Eucalyptus breeding lines, especially the interspecific hybrids. Here, we developed an Agrobacterium-mediated transformation method using peeled nodal-stem explants from in vitro-propagated plantlets of a Eucalyptus hybrid. The explant was prepared by peeling off epidermal layers around the axillary buds using a pair of forceps, before co-cultivation with Agrobacterium tumefaciens. Transgenic shoots generated from the axillary buds were then obtained through kanamycin selection. The transformation efficiency was at most 24% as confirmed by PCR and GUS assay. Transformation of a E. camaldulensis x E. tereticornis F1 hybrid clone using yeast HAL2 showed that, out of 35, 11 transgenic plantlets had an increase in salt tolerance and grew and developed roots, though at a slow rate, on the medium supplemented with NaCl up to 300 mM concentration. This work demonstrates the efficiency of this transformation method for further genetic improvement of Eucalyptus clones selected through hybridization and breeding.