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The aromatic plant Hedeoma multiflora Beth. is endangered by both the overexploitation and the advance of agricultural and urban borders. We report an efficient protocol for its in vitro propagation without using plant growth regulators (PGRs). Two in vitro plant lines, one initiated from seeds and other from binodal segments, remain stable and present high multiplication rates in half strength Murashigue and Skoog medium. Neither the addition of 1-naphthylacetic acid, kinetin or their combinations improved either growing or multiplication rates which reached 26.39 stems per initial explant after 12 weeks. A comparison toward three wild populations, harvested at both early and late summer time, shows that the morpho-anatomical characteristics of the aerial parts are fairly preserved although in vitro plants display more abundant but shorter roots. The profiles of volatile compounds from wild and in vitro specimens are quite similar, although hydrocarbon monoterpenes’ fraction is more important in the latter. Pulegone is by far the main volatile metabolite in all the assessed populations while its proportion drops in the in vitro and in the wild plants from late summer respect to wild plants from December. The concentration of the biomarkers, menthone, isomenthone, pulegone and isopulegone, is about an order of magnitude lower in the in vitro than in the wild plants. Overall, the biomass production in a PGR-free medium ensures constant composition of specialised metabolites and absence of biological and chemical contaminations, representing an alternative for the sustainable use of H. multiflora as edible material.

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