Development of a robust in vitro propagation protocol, assessment of genetic fidelity, and metabolite analysis of Ajuga parviflora Benth
摘要
Ajuga parviflora Benth. (Lamiaceae), commonly known as bugleweed, holds traditional significance due to several biological activities for treatment of fever, hyperthyroidism, premenstrual syndrome, bleeding, diarrhea, obesity, and diabetes. A highly effective in vitro direct and indirect organogenesis protocol using leaf, petiole, and root as explant source has been established. For direct organogenesis, explants were inoculated on MS medium containing 0.025 to 1.0 mg L−1 NAA, 0.05 to 2.0 mg L−1 TDZ, 0 to 2.0 mg L−1 kinetin, and 0 to 2.0 mg L−1 BAP. For indirect organogenesis, explants were inoculated on MS medium containing 0.5 to 2.0 mg L−1 BAP, 0.025 to 1.0 mg L−1 NAA, 0.05 to 2.0 mg L−1 TDZ, 0.5 to 2.0 mg L−1 kinetin, and 0.5 to 4.0 mg L−1 2,4-D. The highest shoot regeneration from callus was attained on MS medium containing 0.25 mg L−1 kinetin and 1.0 mg L−1 BAP. The cloned shoots were accomplished to in vitro rooting with 86.6% with maximum 29.3 healthy roots per plant on MS with 0.5 mg L−1 kinetin and 1.0 mg L−1 IBA. Additionally, 0.2 mg L−1 TDZ and 0.05 mg L−1 NAA supplemented medium exhibited almost 100% regeneration via direct propagation method. Leaf explants were most effective for indirect micropropagation, followed by the petiole, and root. Well rooted plantlets were hardened in a greenhouse using a sand:soil:vermiculite (1:1:1, v/v) mixture under controlled conditions. Genetic uniformity of micropropagated plants was validated through RAPD, SCoT, and ISSR markers. UPLC-PDA and UHPLC-Q-TOF/IMS analysis also revealed that the tissue culture–developed plants maintained metabolic fidelity compared to the mother plant. Consequently, the in vitro propagation protocol of A. parviflora is highly reproducible and can be effectively utilized for the industrial production of valuable bioactive compounds for medicinal and pharmaceutical applications.