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Alkaloids are the main active ingredients in the medicinal plant Dendrobium officinale. Based on the published genomic and transcriptomic data, a proposed terpenoid indole alkaloid (TIA) biosynthesis pathway may be present in D. officinale. In this study, protocorm-like bodies (PLBs) with a high-yielding production of alkaloids were obtained by the optimization of tryptophan, secologanin and methyl jasmonate (MeJA) treatment. The results showed that the total alkaloid content was 2.05 times greater than that of the control group when the PLBs were fed with 9 μM tryptophan, 6 μM secologanin and 100 μM MeJA after 36 days. HPLC analysis showed that strictosidine synthase (STR) activity also increased in the treated plants. A total of 78 metabolites were identified using gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-mass spectrometry (LC-MS) methods; 29 differential metabolites were identified according to the multivariate statistical analysis. Among them, carapanaubine, a kind of TIA, exhibited dramatically increased levels. In addition, a possible underlying process of the metabolic flux from related metabolism to the TIA biosynthetic pathway was enhanced. These results provide a comprehensive view of the metabolic changes related to alkaloid biosynthesis, especially TIA biosynthesis, in response to tryptophan, secologanin and MeJA treatment. [ABSTRACT FROM AUTHOR]