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Clinopodium pulegium (Rochel) Bräuchler (Lamiaceae) is an endangered species endemic to the Southern Carpathians. It is characterized by the production of high amounts of essential oils, which emit volatile organic compounds (VOCs) that have an essential role in biotic and abiotic stress responses and in plant-plant and plant-insect interactions. The present study was initiated to phytochemically examine the influence of different carbon sources in the nutrition medium on VOC emissions of micropropagated C. pulegium plants, using gas chromatography-mass spectrometry analysis of headspace VOCs. The volatile profiles were subjected to multivariate analysis with respect to the presence, concentration and type of carbon source in the nutrient medium. In addition, the effect of different carbohydrates on the density and size of the leaf glandular trichomes, the main structures involved in the emission of VOCs, was determined. A total of 19 VOCs, primarily belonging to mono- and sesquiterpenes previously described in plants, were tentatively identified. Six VOCs were produced at levels higher than 2% of the total VOC emission, dominated by pulegone, ß-pinene and menthone. Inclusion of the carbohydrates in the culture media affected the production of the main leaf trichome-associated volatile allelochemicals although the qualitative composition of the volatiles changed only slightly. Multivariate analysis showed that the concentration, rather than the carbohydrate type, influenced the VOC profile.

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MAIN CONCLUSION: In vitro culture conditions and kinetin induced quantitative modifications in the production of the major volatile constituents in Micromeria croatica plantlets. Antimicrobial activity of methanolic extracts obtained from micropropagated and wild-growing plants was evaluated. Micromeria spp. are aromatic plants, many of which were shown to exhibit various biological effects. The present study aimed to determine the content and the composition of the essential oil of in vitro-cultured Micromeria croatica (Pers.) Schott and to evaluate the in vitro antimicrobial activity of its methanolic extract, in order to compare its phytochemical profile and biological activity with wild-growing plants. Shoots regenerated on MS medium without plant growth regulators (PGRs) or supplemented with kinetin were used for phytochemical analysis. Essential oils from both native plant material and in vitro-cultivated M. croatica plants, with a total of 44 identified constituents, were dominated by oxygenated monoterpenes. Borneol was the main component detected in wild-growing plants (25.28%) and micropropagated plants grown on PGR-free medium (20.30%). Kinetin treatment led to increased oil yield and favored the production of oxygenated monoterpenes, dominated by geranial (33.53%) and cis-p-mentha-1(7),8-dien-ol (23.69%). The percentage of total sesquiterpenoids in micropropagated plant material was considerably lower than in wild-growing plants. In vitro culture conditions and PGRs affected the production of essential oils, inducing quantitative modifications in the production of the major volatile constituents in M. croatica plantlets. The antimicrobial activity of M. croatica methanolic extracts was investigated using the broth microdilution method. Extracts obtained from in vitro cultures generally exhibited greater antibacterial potential, compared to wild-growing plants. Among six bacterial strains tested, Bacillus cereus and Staphylococcus aureus were the most sensitive microorganisms. The present study provided evidence that in vitro culture conditions might favorably affect the antimicrobial activity of M. croatica methanolic extracts.

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The anatomy and ultrastructure of the short glandular trichomes occurring on young expanding leaves of Nicotiana tabacum were investigated using light and transmission electron microscopy. The objective of the present research was to characterize the cellular changes that occur during morphogenesis of short glandular trichomes, from initiation to senescence. Ultrastructural analysis of their secretory cells revealed characteristics common to gland cells: numerous mitochondria in highly organized cytoplasm, large nuclei, and an elaborate network of endoplasmic reticulum. Initial changes in nuclear and plastidial organization were observed at a more advanced secretory stage, marking the onset of senescence. During trichome senescence, gradual reduction of the cytoplasm density occurred along with structural changes of the plastids and the tonoplast. As a result of inward blebbing of the cytoplasm into the vacuole, membrane bound vesicular structures appeared in the vacuolar space. At the late secretory stage, marked by an increase in vacuolation and extraplasmic space, degenerative changes included further fragmentation of the cytoplasm and deterioration of the tonoplast. Multimembrane myelin bodies observed in the vacuolar space were indicative of membrane digestion although plasma membrane did not appear massively degraded.

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MAIN CONCLUSION: In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N(6)-benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 µM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.

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