In vitro micropropagation and conservation of endangered medicinal plant Nepeta asterotricha Rech.f. (Lamiaceae): genetic fidelity, phytochemical and biological assessment

An efficient in vitro protocol was introduced for the conservation of Nepeta asterotricha, a vulnerable and endangered medicinal species found in the central of Iran for the first time. Growth, phytochemical, and biological traits of in vitro regenerated plant (RP) and acclimated plant (AP) were compared to the mother plant (MP). In addition, the genetic stability of AP was assessed by using inter-simple sequence repeats (ISSR) markers. The highest number of lateral branches (4.25) was obtained from the medium with 3 mg/mL kinetin (KIN), while the highest length of lateral branches (13.25 cm) was achieved on the medium culture fortified with 3 mg/mL thidiazuron (TDZ) and 6-benzylaminopurine (BAP). The highest number of leaves (20.25) and main branch length (12.25 cm) were obtained from the medium containing 3 mg/mL TDZ. The highest number of roots (46.25) and root length (2.25 cm) was measured from the medium fortified with 1 mg/mL indole-3-butyric acid (IBA) and 0.6 mg/mL indole-3-acetic acid (IAA), respectively. RP was successfully acclimated (85%) in vivo. Molecular analysis showed that the AP was true to the type of the MP. cis-Sabinene hydrate (26.8-57.7), 1,8-cineole (6.2-24.1), 4a alpha,7 beta,7a alpha-nepetalactone (4.1-12.3), and terpinene-4-ol (3.2-15.0) were the major essential oils compounds. The studied samples contained rosmarinic acid (2.55-5.97 mg/g DW), cichoric acid (1.68-12.7 mg/g DW), chlorogenic acid (1.91-64.21 mg/g DW), rutin (0.59-1.09 mg/g DW), apigenin (0.52-0.72 mg/g DW), betulinic acid (0.17-2.20 mg g DW), oleanolic acid (0.84-5.37 mg/g DW) and ursolic acid (3.46-15.70 mg/g DW). Acclimated plant exhibited the highest antioxidant activity (IC50 = 196.4 mu g/mL), while the methanolic extract of MP displayed the highest antibacterial activity (MIC = 8 mg/mL) against Staphylococcus aureus.