Sunflower in vitro culture regeneration is highly dependent on the genotypic context and culture conditions. However, there is insufficient information about the effect of the cytoplasm on the regeneration ability of this species. The aim of this experiment was to evaluate the effect of different cytoplasmic background on the regeneration ability of HA89, a well known recalcitrant inbred line. Cotyledons of seven HA89-alloplasmic lines and the fertile HA89 inbred line were used as explants on four different culture media. After 36 days of incubation, the regeneration percentage, as well as the productivity and proliferation rates, was recorded. Genotype × culture media interaction was detected in order to obtain regeneration percentage. Further analysis of this interaction demonstrated that the effect of the cytoplasm and the resulting interactions with the culture medium are key factors to be taken into account for better understanding of the in vitro culture performance of this species. Furthermore, non-nuclear genome could be considered as another source of variability modifying the regeneration ability of recalcitrant sunflower genotypes.