Micropropagation and Genetic Fidelity of Rheum Ribes L.: A Threatened and Endemic Medicinal Herb from the Turkish Eastern Anatolia Region

Abstract

Rheum ribes L., an endemic medicinal herb of Eastern Anatolia, faces serious conservation concerns due to habitat degradation, seed dormancy, and overharvesting. To overcome these challenges, a robust and reproducible in vitro regeneration system was developed using embryo axis, hypocotyl, and cotyledon explants via direct and indirect organogenesis as well as somatic embryogenesis. The highest germination rate (88.8%) and shortest germination time were obtained using 1/2 MS medium supplemented with 1.75 mg/L GA(3) and 1.0 mg/L IBA in mature embryos. The micropropagation experiments were conducted in two stages. In the first stage, embryo axis, cotyledon, and hypocotyl explants were cultured using 36 different methods that included various culture conditions and types of plant growth regulators at differing concentrations. In the second stage, calli from these explants were cultured in the most effective medium (method 30) for callus production, and the explants were subsequently cultured in eight different regeneration media based on MS medium supplemented with varying concentrations of plant growth regulators. Callus induction reached 100% under dark conditions in MS medium containing 0.5 mg/L BAP, 0.5 mg/L KIN, and 1.0 mg/L 2,4-D. Among 36 regeneration protocols, method 13 (3.0 mg/L BAP, 0.2 mg/L NAA, 1.0 mg/L GA(3)) yielded the highest plant regeneration (1.15 plantlets/explant) in embryo axis and hypocotyl explants. In the second stage, the highest indirect somatic embryogenesis (ISER) rate (55.6%) was observed in cotyledon-derived callus (C-callus). In contrast, the highest indirect organogenesis (IOR) (28.1%) was found in embryo axis-derived callus (E-callus). The maximum number of plantlets per explant (PPEN: 1.08) was achieved in MS medium containing 2.0 mg/L BAP, 2.0 mg/L KIN, and 1.0 mg/L GA(3). Genetic fidelity of 15 regeneration systems was assessed using 20 highly polymorphic SCoT primers. Similarity coefficients between regenerated plants and the mother plant ranged from 0.904 to 0.991, with the highest genetic stability observed in plantlets derived via direct somatic embryogenesis. This optimized regeneration protocol is a powerful tool for the large-scale clonal propagation and ex-situ conservation of R. ribes, enabling both genetic preservation and sustainable biotechnological utilization of this pharmacologically valuable species.