An efficient micropropagation protocol with anatomy and genetic homogeneity assessment of Calligonum polygonoides Linn.: an endangered and woody species of Rajasthan
摘要
Calligonum polygonoides is a woody, endangered, and keystone species of the Indian Thar desert. The present study is the first report that represents an efficient and rapid micropropagation protocol for this species. Nodal shoot segments of intermediate diameter from mature plants harvested during the spring season were found to be best for the establishment of aseptic culture. Approximately 80% of the explants exhibited an in vitro axillary bud break on Murashige and Skoog (MS) medium fortified with 3.0 mg/L 6-benzyleaminopurine (BAP). The shoot proliferation frequency was enhanced by optimizing successive transfers of mother explant, nutritional media, type, and concentration of exogenously applied plant growth mediators (PGRs). The best shoot multiplication (115.33 ± 10.10) and average length (7.57 ± 0.52 cm) were observed on modified murashige and skoog medium (MMS) medium that had double-strength macro salts (CaCl2.2H2O, KH2PO4, and MgSO4.7H2O) and was supplemented with 0.25 mg/L BAP, 0.25 mg/L kinetin (KN), and 0.025 mg/L indole-3-acetic acid (IAA). The in vitro regenerated shoots were rooted on full strength of MS medium fortified with 0.1 mg/L indole-3-butyric acid (IBA). The comparative analysis of stem anatomy revealed the differentiation of mechanical tissue development under in vitro conditions, which remarks on the survivability of plantlets in the natural environment. Further, the validation of genetic homogeneity using Start codon targeted and inter-simple sequence repeat markers revealed 95.93% monomorphism across all 15 micropropagated plants as well as the mother plant. The higher value monomorphic banding pattern demonstrates the reliability of this micropropagation system for C. polygonoides. Hence, the discussed protocol can be applied for large-scale multiplication and conservation of elite genotypes.