Sunflower (Helianthus annuus L.) is considered one of the recalcitrant species in terms of transformation and regeneration. A routine transformation system of this crop requires competent cell cultures for efficient plant regeneration as well as an effective method for gene delivery. A transformation system was developed by an Agrobacterium tumefaciens-mediated method using split mature embryonic axis explants from the Ha89 genotype. Mean transformation efficiency obtained (measured as PCR+ plants/treated explants) varied from 1 to 5.2% depending on the use of the EHA105 or the C58 strain containing a plasmid with a gene of agronomic interest. The system developed has applicability to several Agrobacterium strains and plasmids with both reporter genes or genes of agronomic interest. Plants obtained with this protocol were confirmed by PCR and Southern blot. Stable inheritance of transgenes was successfully followed until generation T4 in several independent lines.