Genetic transformation of Castanea sativa Mill. by Agrobacterium tumefaciens

Cotyledonary node explants excised from embryonic axes and globular or early-torpedo somatic embryos of Castanea sativa Mill. were used as explants for the genetic transformation of chestnut. Murashige and Skoog (MS) medium containing 150 mg/L of kanamycin was used as selecting medium. The best transformation efficiency was achieved when somatic embryos were co-cultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes. The addition of acetosyringone did not improve the transformation efficiency, which was highest when somatic embryos were infected with the bacterial culture in the exponential phase of growth (OD600 = 0.6). Co-culture of the cotyledonary node explants with different Agrobacterium strains resulted in a lower transformation rate. The best results were achieved when embryonic axes were precultured on medium with TDZ, and the excised nodal explants were co-cultured for 5 days with the EHA105 pUbiGUSINT strain.