Molecular Breeding of Eucalyptus grandis: Developing an Efficient Transformation Method

Eucalyptus grandis is one the most widely planted species in forests in Tropical and Sub Tropical regions. The development of an efficient transformation protocol may support the improvement of Eucalyptus resources through functional genomics and biotechnology. The need for a reliable model for the functional analysis of genes still exists, namely when the research purpose aims to assess if a selected candidate gene may interfere with different wood quality aspects and technological features. However, the prerequisites for the success of the transformation strategy are the establishment of an efficient in vitro regeneration and the optimization of a selection scheme to recover transgenic plants.

The main purpose of this work was to develop an efficient regeneration- transformation method for Eucalyptus grandis. Different types of explants and growth conditions were tested, including different plant growth regulator supplementations and media. Microprogation was enhanced when shoots were cultivates in MS medium supplemented with 0.5 mg/l Zea and 0.2 mg/l NAA. Shoot organogenesis was achieved when leaf explants were cultured in 1.0 mg/l Zea plus 0.1 mg/l NAA in MS medium. The adequate kanamycin concentration to be used for selecting transgenic E.grandis tissues was also tested by inducing regeneration under increasing concentrations of this antibiotic (10, 15, 20, 25, 50 & 100mg/L). Preliminary Agrobacterium tumefaciens mediated transformation experiments were made using the pMP2482 vector with a fusion of GUS:int/GFP, under the control of CaMV35S promoter. 

An important output of this study was the confirmation of a seasonal effect on the shoot regeneration ability of E. grandis.

Acknowledgements:

This work has been partially supported by Fundação para a Ciência e Tecnologia (Portugal) throughout the projects microEGo (PTDC/AGR-GPL/098179/ 2008) and TREEFORJOULES (P-KBBE/AGR-GPL/0001/2010). SSA and JAPP acknowledge their research contracts by the Ciência 2008 program.