BackgroundTo establish an indirect regeneration protocol inFicus lyrata, a three-phase experiment (callus induction, morphogenic callus induction, and plant regeneration) based on auxin, cytokinin, and nitric oxide interactions was designed and implemented using leaf explants. The metabolite profiles (amino acid profile, total phenolic content, total soluble sugars, and total antioxidant activity) alteration patterns were also investigated to determine the metabolites contributing to the progress of each phase.ResultsResults demonstrated that 11 out of 48 implemented treatments resulted in morphogenic callus induction (morphogenic treatments), and nitric oxide played a key role in increasing efficiency from 13 to 100%. More importantly, nitric oxide cross-talk with cytokinins was necessary for shoot regeneration from morphogenic calli. Only 4 out of all 48 implemented treatments were capable of shoot regeneration (regenerative treatments), and among them, PR42 treatment led to the highest shoot regeneration rate (86%) and maximum mean number of shoot/explant (10.46). Metabolite analyses revealed that the morphogenic and regenerative treatments followed similar metabolite alterations, which were associated with increased biosynthesis of arginine, lysine, methionine, asparagine, glutamine, histidine, threonine, leucine, glycine, serine amino acids, total soluble sugars content, and total antioxidant activity. On the contrary, non-morphogenic and non-regenerative treatments caused the accumulation of a significantly greater total phenolic content and malondialdehyde in the explant cells, which reflexed the stressful condition of the explants.ConclusionsIt could be concluded that the proper interactions of auxin, cytokinins, and nitric oxide could result in metabolite biosynthesis alterations, leading to triggering cell proliferation, morphogenic center formation, and shoot regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04339-z.