Introduction of the RS-AFP2 gene into Eucalyptus urophylla for resistance to Phytophthora capsici

We developed an Agrobacterium tumefaciens-mediated transformation system for Eucalyptus urophylla using hypocotyl explants. Antibiotic concentrations, pre-culture times, pH of the inoculation medium and co-culture times were optimised. Pre-cultured hypocotyl explants were co-cultured with A. tumefaciens strain EHA105 harbouring the binary vector pPBR-2 containing the Rs-AFP2 gene, which encodes an antifungal protein, under the control of the prp1-1 promoter, for six days and were then transferred to selective callogenesis-inducing medium containing kanamycin and cefotaxime. Calluses developed shoots and were cultured in an elongation medium and finally multiplied. The integration of T-DNA into the genome of transgenic E. urophylla was confirmed by polymerase chain reaction (PCR). The reverse transcription (RT)-PCR results showed that Rs-AFP2 gene expression could be detected only after the transformed plants were inoculated with Phytophthora capsici 60 hours after inoculation. These results indicated that the prp1-1 promoter was inducible and Rs-AFP2 could enhance the resistance of E. urophylla to P. capsici. This protocol enabled effective transformation and regeneration of E. urophylla.