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Abstract The simultaneous and sequential dual elicitation effect on plumbagin production in Plumbago indica L. root cultures, revealed that combination of chitosan (150 mg L−1) with ʟ-alanine (5 mM) or methyl-β-cyclodextrin (MCD; 2 mM) significantly increased plumbagin production, but in the different treatment manners. The simultaneous treatment using chitosan + ʟ-alanine on a 14-day-old culture enhanced plumbagin production to 14.62 mg g−1 DW, while the sequential additions of MCD to a 12-day-old culture followed by chitosan after 48 h enhanced production of plumbagin to 14.33 mg g−1 DW. The plumbagin productivity from both treatments were up to 1.3- and 8-fold higher than the chitosan treated (10.93 mg g−1 DW) and untreated root cultures (1.76 mg g−1 DW). Moreover, the present studies provided new information on the effect of simultaneous and sequential elicitation on plumbagin-producing P. indica root cultures using chitosan in combinations with MCD or ʟ-alanine.