The in vitro rooting of three caper (Capparis spinosa L.) selected biotypes, grown in a commercial orchard on the Sicilian island of Salina (38°33′49” N), was performed using—as base material for rooting experiments—shoot explants proceeding from two different in vitro culture systems: solid medium and liquid culture in a PlantForm bioreactor (TIS). The regenerated shoots of each accession were submitted to different auxin treatments (NAA, IBA, IAA - 1 or 2 mg L−1; NAA+IBA 0.75 and 0.25 mg L−1, respectively), supplemented with sucrose or fructose (mg L−1). The highest rooting rate in terms of root percentage (67%) was reached with the explants of the selected accession ‘Sal 39’ proceeding from liquid culture in PlantForm and induced in the MS medium with sucrose, as a carbon source, supplemented with NAA 0.75 mg L−1 + IBA 0.25 mg L−1, after six days in a climatic growth chamber at 25 ± 1 °C in the dark and then placed under a cool white fluorescent lamp, with a PPFD of 35 μmol m−1 s−1 and a photoperiod of 16 h. On the other hand, poor rooting rate was generally achieved under all the tested experimental conditions with the other biotypes, ‘Sal 37’ and ‘Sal 35’, demonstrating the strong role exerted by the previously adopted proliferation method and by the genotype for successful caper in vitro rooting.