Helianthemum guttatum Mill and Helianthemum ledifolium are herbaceous Cistacea species, known for their drought tolerance and symbiotic association with desert truffles (Terfezia, Tirmania…). Even if their propagation mainly occurs through seeds. However, germination is limited by integument-induced physical dormancy. Micropropagation may provide an effective alternative for overcoming this constraint. The present study aimed to developan in vitro regeneration method to facilitate large-scale production of host plants for sustainable truffle cultivation. Seeds collected from native Moroccan populations were subjected to mechanical scarification to enhance germination. Germination and shoot proliferation of axillary and apical buds were evaluated on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP: 0, 0.5, 1 and 2 mg.L-1). A maximum germination rate of 100% was observed in scarified seeds cultured on MS or agar media. Shoot proliferation was highest in BAP-free medium, reaching 100% and 95% regeneration from axillary and apical buds, respectively. Root induction was also optimal in the absence of BAP, with 100% for both species. Higher BAP concentration (1-2mg.L-1) promoted callus formation at the expense of shoot elongation and rooting. Acclimatized plantlets showed survival rates of 90% for H.ledifolium and 100% for H.guttatum. This optimized protocol provides a reliable system for large-scale propagation of Helianthemum species, ensuring a sustainable supply of host plants for desert truffle inoculation in arid and semi- arid Mediterranean regions.