Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.
In this study, tansy (Tanacetum vulgare L.) in vitro culture was established from seeds collected from natural populations. The multiplication of plantlets was conducted through shoot tips that exhibited potent apical growth and regeneration capacities on basal medium (BM), without the addition of any plant growth regulators (PGRs). PGRs were also omitted for the establishment and cultivation of tansy root cultures. Both abaxial and adaxial leaf surfaces of in vitro micropropagated plantlets were covered with glandular biseriate trichomes. Histochemical staining showed that glandular secretions were rich in lipid and terpene compounds, confirmed by GC-MS analysis of essential oil (EO). In the total EO, similar portions of oxygenated monoterpenes (38.5% m/m) and oxygenated sesquiterpenes (22.6% m/m) were detected. Chemical profiles of methanol extracts of in vitro cultured tansy shoots and roots varied in quantity and quality from those obtained from wild-growingtansy. HPLC analysis indicated that the methanol extracts of in vitro cultured roots were the richest in 3,5-O-dicaffeoylquinic acid (3,5-O-DCQA), in which the concentration was 6 times higher (10.220 mg/g DW) than that in the extract obtained from roots of wild-growing tansy (1.684 mg/g DW). This result is noticeable in the manner of industrial production of biologically active 3,5-O-DCQA that has been shown to have antioxidant, hepatoprotective, antiviral, antimutagenic, and immunomodulatory activity. Biotechnological interventions on secondary metabolite production taking place in trichomes could further enhance the production of some important tansy metabolites and further investigation will be directed toward the elucidation of the pharmaceutical potential of tansy in vitro obtained metabolites, as mixtures or single moieties.
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.
In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 µM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.
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