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Effectiveness in woody biomass utilization is highly dependent on its genetics and physiology. We performed morpho-anatomical, chemical, and biomethane productivity characterizations of one-year-old woody stems in three shrub Salix viminalis genotypes: a diploid (Energo) and its two autotetraploid derivatives (PP-E7 and PP-E13). Tetraploidization affected changes in stem morpho-anatomy and corresponding improved chemical features and biomethane productivity, considerably more pronounced in tetraploid PP-E13, while PP-E7 was more similar to diploid Energo. Compared to diploid Energo, in tetraploid PP-E13 morphometric analysis showed increased stem diameter and higher wood fiber radial double wall thickness, while microscopic analysis suggested higher syringyl to guaiacyl (S:G) ratio of the wood fiber cell wall. Presented changes in stem morpho-anatomy of tetraploid PP-E13 compared to diploid Energo correspond to the improved chemical features: the lower Klason lignin content and higher S:G ratio, the higher cellulose and xylan content, and lower cellulose crystallinity (Crl). Presented improved chemical features, along with the increase in ash content, resulted in a 7.3% (10.3 CH 4 mL/g VS) increase in biomethane productivity in tetraploid PP-E13, compared to diploid Energo, suggesting tetraploid PP-E13 as an optimal raw material for fermentation technologies. In addition, besides the well-known chemical markers of willow biomass quality, the presented results highlight key stem morpho-anatomical parameters, which can serve as additional markers in energy willow improvement.

Anatomical adaptation of liana plants includes structural changes in cell walls of different tissues: fibers, vessel elements and tracheids. However, the contribution of parenchyma cells to stem twining in liana plants is mostly unknown. The aim of this investigation is to determine changes in stem parenchyma cell walls that are correlated with the twinning process in liana plants. Parenchyma cell wall structure was studied on the stem cross sections of straight and twisted internodes of monocotyledonous liana Dioscorea balcanica, by different microscopy techniques: light microscopy, scanning electron microscopy, fluorescence detected linear dichroism microscopy and Fourier transform infrared microspectrometry. In addition, chemical analysis of the entire stem internodes was performed using photometric and chromatographic methods. Parenchyma cell walls of twisted D. balcanica internodes are characterized by: lower amounts of cellulose (obtained by FTIR microspectrometry) with different cellulose microfibril orientation (shown by Scanning electron microscopy), but no changes in \"cellulose fibril order\" (obtained by Differential polarization laser scanning microscopy); lower amounts of xyloglucan, higher amounts of xylan, higher amounts of lignin with modified organization-less condensed lignin (obtained by FTIR microspectrometry). At the same time, chemical analysis of the entire internodes did not show significant differences in lignin content and cell wall bound phenols related to stem twining, except for the presence of diferulate cross-links exclusively in twisted internodes. Our results indicate that adaptations to mechanical strain in D. balcanica stems involve modifications in parenchyma cell wall structure and chemistry, which provide decreased stiffness, higher strength and increased elasticity of twisted internodes.

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.

Gibberellins (GAs) play a pivotal role in the induction of somatic embryogenesis from in vitro root apices of spinach plants. With the aim to understand the role of GAs in this process and to improve somatic embryo (SE) regeneration efficiency, the impact of light and GAs on SE initiation from the in vitro root apices was studied. The root sections were isolated from in vitro-grown SE-derived plants and placed on medium containing 20 µM α-naphthaleneacetic acid (NAA) and 0–10 µM GA3 or GA1, and cultivated under light conditions or in darkness. The most efficient SE regeneration response (100% regenerating SEs and 40.73 SEs per root apices) was achieved only in the presence of both light and GAs, with GA3 always exhibiting much stronger effect than GA1. Considering that light enhances GAs biosynthesis and the necessity of GAs for SE initiation, the expression levels of genes encoding the key enzymes involved in the final steps of GAs synthesis (SoGA20-ox1 and SoGA3-ox1) and deactivation (SoGA2-ox1, SoGA2-ox2 and SoGA2-ox3) were analyzed. Light enhanced the expression of all five GA-ox genes, while exogenously supplied NAA + GA3 provoked downregulation of SoGA20-ox1 and SoGA3-ox1 and upregulation of SoGA2ox-2 and SoGA2ox-3 expression. The expression of SoGA2ox-1 only slightly decreased. The results indicated the capability of isolated spinach roots to perceive the light and autonomously produce GAs. The expression levels of genes encoding key enzymes involved in GA biosynthesis suggest that lower levels of GAs favor SE initiation.Key messageLight and gibberellins (GA) synergistically promote somatic embryogenesis in spinach. Expression levels of genes encoding key enzymes for GA metabolism suggest that lower levels of GAs may favor somatic embryogenesis.

The present study evaluates the phytotoxic effects of phloretin, a prevalent secondary metabolite of apple trees, on the broadleaf weed Capsella bursa-pastoris (L.) Medik. known for its resistant myxospermous seeds that form a long-lasting soil bank. The results indicate a significant, dose-dependent inhibitory effect of phloretin on the growth and morphological parameters of weed seedlings grown in vitro. Although the applied phloretin concentrations (250–1000 µM) were not lethal to the C. bursa-pastoris seedlings after two weeks, the metabolism of the seedlings was impaired, resulting in an accumulation of lipid droplets in the root tips and root hairs. Histochemical analysis shows deposits of phenols in the root epidermal cells, which are probably aggregates of phloretin or its metabolic derivatives. The accumulation of pectin in the cell walls of root border cells in phloretin-treated seedlings indicates an attempt to reduce the uptake of phloretin and reduce its concentration in the cells. Inhibition of shoot growth associated with chlorosis and reduced photosynthetic pigment content is a consequence of seedling exposure to phloretin. This study provides a basis for further evaluation of phloretin as a new bioherbicidal compound and for elucidating the mechanism underlying its phytotoxic activity.

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 N⁶–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 the original publication of the article, one of the project numbers was omitted in the Acknowledgments. The correct version is provided below.

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.

Unlike garlic and onion, the regeneration of chive (Allium schoenoprasum L.), cultivated both for culinary and ornamental purposes, has not been intensively studied. The effects of the eight cytokinins and the plant basal section thickness on regeneration efficiency and subsequent plant growth were studied. Representatives of all cytokinin structural groups: isoprenoide side chain (trans-zeatin) and aromatic side chain (benziladenine, kinetin, meta-topolin) adenine derivatives, and phenylurea derivatives (thidiazuron and N-(2-chloro-4-pyridyl)-N′-phenylurea) at 0, 1, 5 or 10 μM were used. Histological analysis revealed adventitious buds formation from the leaf sheaths’ bases and the basal plate. The highest regeneration frequency (100 %) and the mean bud number per explant (20.0) were achieved with 10 μM thidiazuron (TDZ), and 5 mm-thick basal sections were the most responsive explants. Inferior shoot and root growth characteristics of plants regenerated by this treatment was avoided by exclusion or replacement of 10 μM TDZ with 5 μM kinetin (Kin) after a 4-week bud induction period, without consequences on the regeneration efficiency. In addition, a positive correlation between peroxidase, catalase and superoxide dismutase activity and the regeneration capacity was observed. All antioxidative enzymes activity changed much faster with 10 μM TDZ than with 1 μM Kin, which provoked the weakest regeneration response. Moreover, a unique peroxidase isoform was observed only in TDZ-treated explants after 3rd day of treatment. This work is useful for genetic engineering and virus-free plant production advancement, and for the knowledge expansion regarding the role of antioxidative enzymes in plant organogenesis.

The effect of reduced availability of sugars on growth and essential metabolic processes in roots, resulting from decreased photosynthesis under salinity, was excluded by establishing a non-photosynthetic model-system in this study: root cultures of Centaurium maritimum (L.) Fritch and Centaurium spicatum (L.) Fritch. The contribution of inorganic cations and organic compounds (e.g. carbohydrates and amino acids) to the osmotic adjustment (OA) in roots during short-term exposure to various salt concentrations (0, 50, 100 or 200 mM NaCl) was emphasized. Observed morphological and histological changes in roots were species specific, and were dependent on salinity level. Although C. spicatum appears to be more tolerant to salt stress, both species employed similar strategies in response to elevated salinity to different extents, and displayed effective OA mechanisms. Under low and moderate salinity, inorganic cations were the major contributors to OA in roots of both species, followed by soluble sugars, while the relative contribution of proline (Pro) and free amino acids was insignificant. Osmotic adjustment under severe stress appears to be mediated by increased accumulation of organic compounds. The analysis of the intraspecies variability in salt response of C. spicatum and C. maritimum roots enabled the identification of some organic compounds which could be used as potential biochemical markers in screening for salt tolerance, including Pro in C. spicatum, and trehalose and polyols in C. maritimum.