Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment.However,the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT.This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars.Here,we report that non-GT cells of self-pollinated inbred A.annua plants can express the artemisinin biosynthetic pathway.Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A.annua plants.LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin,artemisinic acid,and arteannuin B.Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A.annua plants.Moreover,we employed all the above approaches to examine artemisinin biosynthesis in the reported A.annua glandless (gl) mutant.The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin.Collectively,these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A.annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.