Abstract
Rhaponticum carthamoides (Willd.) Iljin. (Leuzea carthamoides, Maral root), a medicinally valuable species listed in the Red Book of Kazakhstan, is known for its rich phytochemical profile. However, limited data exist on its microclonal propagation. This study aimed to optimize in vitro and medium-term storage conditions using biotechnological methods. Mature seeds collected from natural populations in the Kazakhstani Altai were germinated, and tissues from the seedlings were used as explants. Sterile shoots were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 mg L−1 −6-benzylaminopurine and 3.0 mg L−1 kinetin. For shoot induction, MS medium supplemented with 0.5 mg L−1 meta-Topolin and using stem apices as explants yielded optimal results. Medium-term storage with chlorocholine chloride at 0.1–0.4 g/L effectively preserved regenerative capacity for further rooting. After 12 months of storage, plantlets were transferred to half-strength MS medium with 3.0 g/L activated carbon and at 2.0 or 5.0 mg L−1 indole-3-butyric acid for rooting. Regenerated plants were successfully acclimatized ex vitro. The 20-hydroxyecdysone content in field-grown plants post-storage reached 9.24 mg/mL, 2.4-fold higher than in wild plants. Inter simple sequence repeat analysis confirmed genetic stability. Our optimized protocol ensures high-yield metabolite production and genetic fidelity, enabling in vitro conservation, nursery-scale cultivation, and the restoration of R. carthamoides natural populations.