Sunflower is a crop species well adapted for cultivation in open fields under full sunlight. Young plantlets can be grown in growth chambers under low irradiance, where different aspects of light can be easily tracked. Using time-lapse imaging, we have shown how monochromatic red, blue, and far-red light and their combinations interacted, affecting the rhythmicity and elongation of sunflower hypocotyls. Monochromatic light of any color, applied individually, canceled all manifestations of diurnal rhythmicity and anticipation of imminent light transitions present in diurnal photoperiods established by white LED light panels. Monochromatic light also significantly increased the rate of hypocotyl elongation, which became uniform (arrhythmic) and often triggered the appearance of guttation. The rate of hypocotyl elongation was highest with the blue light and lowest with red light. In double light combinations, red light suppressed the stimulative effect of blue light, but it promoted the elongation rate when used together with far-red light. A triple light combination of red, blue, and far-red light stimulated hypocotyl elongation to a high degree and increased the elongation rate more than twofold compared with red and fourfold compared with white LED light.

Wild rocket (Diplotaxis tenuifolia (L.) DC) is widely cultivated as a vegetable crop known for its nutritional and medicinal benefits, but also for its value as an oil, pasture and beekeeping crop. Biotechnological approaches are essential to respond to the new challenges in breeding, as conventional breeding methods have proven insufficient for further improvements. The aim of this study was to develop a shoot regeneration and propagation protocol for D. tenuifolia and to investigate its metabolic profile. Optimal shoot multiplication was achieved using Murashige and Skoog medium (MS) supplemented with 0.2 mg l-1 6-BA, yielding 3.04 shoots per explant. IBA 0.5 mg l-1 promoted shoot rooting and acclimatization (80 %) in the greenhouse. Somatic embryogenesis was developed for the first time using immature zygotic embryos (IZE) cultured on MS medium without plant growth regulators (PGR-free) with an embryo-forming capacity of 3.3. Histological observation revealed that the somatic embryos (SEs) have unicellular or multicellular origin and passed through the globular, heart, torpedo and cotyledon stages. PGR-free and MS media supplemented with low doses of cytokinins 6-BA or KIN were satisfactory for both SE germination into plants and secondary SE regeneration. SE-derived plants survived efficiently when transplanted into the soil. The entire SE process from IZE isolation to flowering of the acclimatized plants took a total of 24 weeks. LC/MS analysis revealed that in vitro cultured shoots contained a broader spectrum and higher amount of bioactive polyphenols, especially the selected clone SR1 obtained from the super-regenerative IZE. These results can shorten the breeding process through targeted selection, large-scale propagation and maintenance of desirable clones, but also form the basis for breeding through advanced biotechnological strategies.

Daily changes in the content of phytohormones accumulating in the hypocotyls of young sunflower seedlings that grow under a 14/10-h light-to-dark (LD) photoperiod were followed using LC–MS. The objective of the work was to investigate whether the presence of light and light transitions lead to visible changes visible in phytohormone levels on the sixth day after the onset of germination. The LC–MS technique used allowed the simultaneous detection of more than one hundred individual phytohormone derivatives, of which the daily accumulation patterns were determined for nearly fifty members. The daily changes in phytohormone levels followed specific patterns for each phytohormone. There were differences between the daytime and nighttime levels, most likely reflecting the effects of light on phytohormone metabolism. A significant difference was found between cytokinins (CKs) and all other phytohormones, so that CK group of phytohormones may be considered to have a separate role in hypocotyl elongation. Prolonged daytime (postponed dusk) resulted in a rapid disruption of rhythmic hypocotyl elongation and it triggered an acute light stress response that was evident in increased levels of a number of important phytohormones from all groups except CKs. However, this light stress, termed ARELD (Acute Response to Extended Light Duration), was not present in control plants grown under continuous light conditions. The data suggest that sunflower has means to adapt to the potentially stressful conditions of continuous light (LL). The qPCR-RT study of the isoforms of circadian clock-associated genes HaLHY, HaTOC1, HaELF3, and HaPIF3 revealed diurnal rhythms under both LD and LL conditions in which the peaks were synchronized and shifted toward the subjective dawn.

Despite the existence of various therapeutic approaches, diabetes mellitus and its complications have been an increasing burden of mortality and disability globally. Hence, it is necessary to evaluate the efficacy and safety of medicinal plants to support existing drugs in treating diabetes. Xanthones, the main secondary metabolites found in Gentiana dinarica and Gentiana utriculosa, display various biological activities. In in vitro cultured and particularly in genetically transformed G. dinarica and G. utriculosa roots, there is a higher content of xanthones. The aim of this study was to investigate and compare antidiabetic properties of secondary metabolites (extracts) prepared from these two Gentiana species, cultured in vitro and genetically transformed with those collected from nature. We compare HPLC secondary metabolite profiles and the content of the main extract compounds of G. dinarica and G. utriculosa methanol extracts with their ability to scavenge DPPH free radicals and inhibit intestinal α-glucosidase in vitro. Anti-hyperglycemic activity of selected extracts was tested further in vivo on glucose-loaded Wistar rats. Our findings reveal that the most prominent radical scavenging potential and potential to control the rise in glucose level, detected in xanthone-rich extracts, were in direct correlation with an accumulation of xanthones norswertianin and norswertianin-1-O-primeveroside in G. dinarica and decussatin and decussatin-1-O-primeveroside in G. utriculosa.

Eruca sativa is known in traditional medicine for its therapeutic effects, while young plants are used as a salad or green food. Recently, the consumption of rocket has increased considerably, so it has become very important for breeders. Plant tissue culture provides a platform to overcome the problems in improving this species. In the present study, an efficient protocol for in vitro shoot regeneration and propagation of recalcitrant rocket variety Domaća rukola was studied. Murashige and Skoog (MS, 1962) medium containing 0.1 mg L−1 BA and frequent subculture over a period of three weeks proved to be optimal for shoot multiplication with a multiplication index of over 3 and only 8.72% of hyperhydrated shoots without necrosis. Different concentrations of 2,4-D, BA, or TDZ in combination with NAA, with or without the presence of AgNO3, were tested for de novo shoot organogenesis (DNSO) from seedling explants. The hypocotyl explants cultured on MS with a combination of TDZ1.0+NAA0.1+AgNO3 5.0 mg L−1 regenerated viable shoots with the highest rate (25.38%) and an average number of 2.18 shoots per regenerating explant. Somatic embryogenesis from immature zygotic embryos proved to be the best way to regenerate a recalcitrant rocket cultivar. The highest embryogenic efficiency was achieved in explants cultured on MS medium containing 1.0 mg L−1 2,4-D with a frequency of 76.64% and 5.13 mean number of regenerated somatic embryos per explant, which were further converted into normal plants. Additionally, in vitro-produced rocket shoots could serve as a possible promising source for the production of flavonoid kaempferol with proven antioxidant properties.

This study presents the hypocotyl elongation of sunflower seedlings germinated under different light conditions. Elongation was rhythmic under diurnal (LD) photoperiods but uniform (arrhythmic) under free-running conditions of white light (LL) or darkness (DD). On the sixth day after the onset of germination, seedlings were entrained in all diurnal photoperiods. Their hypocotyl elongation was dual, showing different kinetics in daytime and nighttime periods. The daytime elongation peak was around midday and 1–2 h after dusk in the nighttime. Plantlets compensated for the differences in the daytime and nighttime durations and exhibited similar overall elongation rates, centered around the uniform elongation in LL conditions. Thus, plants from diurnal photoperiods and LL could be grouped together as white-light treatments that suppressed hypocotyl elongation. Hypocotyl elongation was significantly higher under DD than under white-light photoperiods. In continuous monochromatic blue, yellow, green, or red light, hypocotyl elongation was also uniform and very high. The treatments with monochromatic light and DD had similar overall elongation rates; thus, they could be grouped together. Compared with white light, monochromatic light promoted hypocotyl elongation. Suppression of hypocotyl elongation and rhythmicity reappeared in some combination with two or more monochromatic light colors. The presence of red light was obligatory for this suppression. Plantlets entrained in diurnal photoperiods readily slipped from rhythmic into uniform elongation if they encountered any kind of free-running conditions. These transitions occurred whenever the anticipated duration of daytime or nighttime was extended more than expected, or when plantlets were exposed to constant monochromatic light. This study revealed significant differences in the development of sunflower plantlets illuminated with monochromatic or white light.

The hairy root clones of Gentiana dinarica cl-B, cl-D, cl-3, and cl-14 were cultivated in parallel in diverse simple bioreactors, including temporary immersion systems RITA® (TIS RITA®), bubble column bioreactors (BCB), and Erlenmeyer flasks (EF), and evaluated for biomass production and xanthone content. The obtained results showed that TIS RITA® and BCB containing ½ MS medium with 4% sucrose provided equally good growth conditions in which the majority of the clones displayed the higher percentage of dry matter (DM%), and xanthones norswertianin-1-O-primeveroside (nor-1-O-prim) and norswertianin production than those cultivated in EF. Thin and well branched hairy root clone cl-B grown in BCB for 7 weeks was superior regarding all growth parameters tested, including growth index (19.97), dry weight (2.88 g), and DM% (25.70%) compared to all other clones. Cl-B cultured in TIS RITA® contained the highest amount of nor-1-O-prim (56.82 mg per vessel). In BCB with constant aeration, cl-B accumulated the highest norswertianin content reaching 18.08 mg/vessel. The optimized conditions for cultivation of selected G. dinarica hairy root clones in highly aerated TIS RITA® and BCB systems contribute to the development of bioreactor technology designed for the large scale commercial production of xanthones nor-1-O-prim and norswertianin.

Flower strips of French Marigold are commonly used pest repellents in potato fields. However, the effect of French Marigold volatiles on potato metabolism, physiology and induced defense is unknown. Thus, a microarray transcriptome analysis was performed to study the effects of French Marigold essential oil (EO) on laboratory-grown potato. After 8 h of exposure to EO, with gas chromatography/mass spectrometry (GC/MS)-detected terpinolene and limonene as dominant compounds, 2796 transcripts were differentially expressed with fold change >2 compared to expression in controls. A slightly higher number of transcripts had suppressed expression (1493 down- vs. 1303 up-regulated). Since transcripts, annotated to different photosynthesis-related processes, were mostly down-regulated, we selected a set of 10 genes involved in the leaf starch metabolism pathway, and validated microarray patterns using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Except for decreased synthesis and induced decomposition of starch granule in leaves, 8 h long EO exposure slightly elevated the accumulation of sucrose compared to glucose and fructose in subjected potato plants. An in vitro feeding bioassay with Colorado potato beetle showed that EO-induced alternations on transcriptional level and in the sugars' metabolism caused the enhancement of feeding behavior and overall development of the tested larvae. Results of comprehensive analysis of transcriptional responses in potato exposed to French Marigold EO provide a basis for further elucidation of molecular mechanisms underlying eco-physiological interactions in companion planting cropping systems.

Research on phototropic (PT) bending in sunflower (Helianthus annuus L. cv. Kondi (Syngenta)) seedling hypocotyls presented herein focused on a comparison of diurnal and free-running photoperiods with the aim of explaining the development of diurnal rhythmicity. PT bending magnitudes and lag phase duration exhibited strong daily rhythmicity in all diurnal photoperiods, contrasting with the uniform PT bending response to constant light (CL) conditions. Plants had a daytime maximum for PT bending magnitudes in experiments starting around midday and a minimum in the dark period in those starting 4 h after dusk. Plants could compensate for large differences in the daytime duration of diurnal photoperiods. They required the first 4 h of darkness to recover and synchronize the PT bending and to start increasing the magnitudes of PT bending. The daily pattern of lag phase duration changes was similar but inverted, showing that synchronization also occurred during nighttime. Darkness was not required for PT bending under CL conditions, however, during diurnal photoperiods it enabled the establishment of diurnal rhythmicity and synchronized changes in PT bending capacity to occur when needed, providing maximal values at midday and minimal during the nighttime. Under prolonged duration of daytime corresponding to the start of CL condition, plantlets rapidly abandoned circadian regulation, their PT bending response becoming arrhythmic.

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L−1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L−1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones—mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside—in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2–3-fold compared to the control shoots. Elicitors at 100–300 μM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Shoot and root in vitro culture of endemic European species Gentiana clusii was established for the first time. The effects of different concentrations of benzyl adenine (BA), 6-phurphurylaminopurine (KIN), indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) on shoot propagation and rooting of G. clusii were investigated. The optimal in vitro conditions for shoot propagation and long-term maintenance were achieved using woody plant medium (WPM) supplemented with 0.5 mg l(-1) KIN, and subsequent application of IBA at 0.5 mg l(-1) significantly improved rooting of these shoots. Root culture was established from excised root tips cultured in Vz. MS liquid media with increasing concentrations of IBA (0.1-1.0 mg l(-1)). A high root growth rate and considerable biomass yield were obtained by addition of 1.0 mg l(-1) IBA. HPLC analysis revealed that in vitro culture considerably promoted the production of secondary metabolites in G. clusii. The selected protocol for shoot propagation (WPM + 0.5 mg l(-1) KIN) increased the content of sweroside, gentiopicrin and norswertianin-1-O-primeveroside (N-1-P) for more than 2-fold compared with the wild plants. IBA promoted N-1-P and norswertianin production in root cultures; their contents were enhanced 6.4- and 18.6-fold, respectively, compared with the wild plants. The extract of these roots displayed the highest antioxidant capacity (IC50 = 66.57 mu g ml(-1)). The established shoot and root propagation protocols facilitate in vitro conservation of G. clusii, and provides a promising tool for the large scale production of valuable secoiridoids and xanthones.

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.

Taking advantage of advanced high performance liquid chromatography–electrospray tandem–mass spectrometry (HPLC–ESI–MS/MS), we screened daily changes in concentrations of endogenous phytohormones of in vitro grown potato (Solanum tuberosum L. cv. Désirée) shoot cultures and checked for possible connections between the rhythmicity of endogenous phytohormones and phototropic bending capacity of the same cultures. Studies done under diurnal 16 h light and 8 h darkness (diurnal) and continuous light (CL) conditions showed prominent daily rhythmicity of endogenous phytohormone levels in both light regimes. Phototropic bending in potato is known to be rhythmic only in the diurnal, whereas in CL conditions the bending response is present but without any daily rhythmicity. For all of the studied phytohormone groups significant differences between the diurnal and CL conditions were found. Changes in the concentration of indole auxins, indole-3-acetic acid (IAA) and its catabolite 2-oxindole-3-acetic acid (OxIAA), were the most prominent. Their levels clearly alternated with level of IAA being high in diurnal and OxIAA in CL conditions. Significant concentration changes were also observed for other phytohormones such as cytokinin ribosides, salicylic acid, abscisic acid and phaseic acid. Observed changes in daily phytohormone levels indicate strong and complex involvement of diverse phytohormone groups in realization of the phototropic bending response of potato shoots.

Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stresstolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerancecruciferous vegetable lines.

Kohlrabi (Brassica oleracea var. gongylodes), with its edible stem tuber formed at the base of the plant stem, presents a valuable source of nutrients. The potential effects of plant growth regulators (PGRs), as well as various concentrations of different sugars on the in vitro development of kohlrabi were studied. Ten-day-old kohlrabi seedlings were cultivated in vitro for 5 weeks at 18?2?C on half-strength MS media containing different concentrations of carbon source such as sucrose, fructose, glucose, xylose and mannitol, combined with or without specific plant growth regulators (N6-benzyladenine (BA), gibberellic acid (GA3), 2,3,5-triiodobenzoic acid (TIBA)). Results showed no tuber formation in all treatments, but growth and development of treated kohlrabi seedlings was significantly affected in a distinctive manner, with a variety of morphological traits being altered in comparison to matching controls.

Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ + 2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC50 = 19.09 µg ml− 1).

BackgroundGlioblastoma multiforme (GMB) is the most malignant of all brain tumors with poor prognosis. Anticancer potential of xanthones, bioactive compounds found in Gentiana dinarica, is well-documented. Transformation of G. dinarica roots with Agrobacterium rhizogenes provides higher xanthones accumulation, which enables better exploitation of these anticancer compounds.Hypothesis/PurposeThe aim of this study was to investigate antiglioma effect of three different G. dinarica extracts: E1—derived from untransformed roots, E2—derived from roots transformed using A. rhizogenes strain A4M70GUS, and E3—derived from roots transformed using A. rhizogenes strain 15834/PI. Further, mechanisms involved in anticancer potential of the most potent extract were examined in detail, and its active component was determined.MethodsThe cell viability was assessed using MTT and crystal violet test. Cell cycle analysis, the expression of differentiation markers, the levels of autophagy, and oxidative stress were analyzed by flow cytometry. Autophagy and related signaling pathways were assessed by immunoblotting.ResultsE3, in contrast to E1 and E2, strongly reduced growth of U251 human glioblastoma cells, triggered cell cycle arrest in G2/M phase, changed cellular morphology, and increased expression of markers of differentiated astrocytes (glial fibrillary acidic protein) and neurons (β-tubulin). E3 stimulated autophagy, as demonstrated by enhanced intracellular acidification, increased microtubule-associated light chain 3B (LC3-I) conversion to autophagosome associated LC3-II, and decreased level of selective autophagy target p62. Induction of autophagy was associated with Akt-dependent inhibition of main autophagy suppressor mammalian target of rapamycin (mTOR). Both genetic and pharmacological inhibition of autophagy suppressed the expression of differentiation markers, but had no effect on cell cycle arrest in E3-treated cells. E3 stimulated oxidative stress, and antioxidants vitamin E and N-acetyl cysteine inhibited autophagy and differentiation of E3-treated U251 cells. The most prevalent compound of E3, xanthone aglycone norswertianin, also arrested glioblastoma cell proliferation in G2/M phase and induced glioblastoma cell differentiation through induction of autophagy and oxidative stress.ConclusionThese results indicate that E3 and its main active component norswertianin may serve as a potential candidate for differentiation therapy of glioblastoma.

Production of innovative drugs from natural products in controlled conditions plays an important role in modern pharmacology in order to tackle global health challenges. The potential of Gentiana utriculosa hairy roots, obtained by transformation with Agrobacterium rhizogenes A4M70GUS, as well as that of the shoots regenerated from them via somatic embryogenesis, for xanthone production was investigated. Gentiana utriculosa was shown to be a new source of xanthones, medicinal raw materials for different pharmaceutical applications, among which decussatin has been recently recognized as a prospective hepatoprotective and antiulcer compound. Decussatin and decussatin-1-O-primeveroside were detected in both hairy roots and transgenic shoots, while mangiferin, present in nontransformed plants, was not detected. Quantitative HPLC analysis revealed up to 4.5-fold higher decussatin production in hairy roots line 9 compared with the plants from the nature, and this clone was selected. It showed stable growth after more than two years of continuous subcultivation. Cultivation of hairy roots on solid medium was favorable for biomass production, while liquid culture was beneficial for decussatin and decussatin-1-O-primeveroside accumulation. Cytokinins kinetin or N-6-benzyladenine promoted somatic embryo maturation and germination as well as multiplication of obtained plantlets, while active charcoal reduced hyperhydricity. Plants regenerated from selected hairy root line 9 comprised two TL-DNA inserts, as confirmed by Southern blot analysis. They accumulated at least 2.5-fold more decussatin then nontransformed plants. Therefore, these plants could be valuable material to create xanthone high-yielding cultivars of G. utriculosa.

This study investigated the role of antioxidant system of Alyssum markgrafii, during long-term exposure to 0.5 or 1 mM NiCl2 × 6H2O in vitro. Applied methodology included sample preparation protocol which reduces oxidation of key metabolites along with novel luminescent method and well-established photometric procedures. During 5-week treatments, plants accumulated 1121 and 2470 ppm of Ni2+ respectively, followed by severe growth retardation, chlorophyll degradation and peroxidation of lipids. These effects were more pronounced after 1 mM Ni2+ treatment and additionally accompanied by increased water loss. Activities of luminol-converting peroxidases and glutathione reductase upon 0.5 mM treatment were increased while catalase and superoxide dismutase were diminished. The fact that these two groups of enzymes run in antiparallel might suggest functional redistribution between antioxidant enzymes rather than orchestrated action to prevent oxidative damage. Total antioxidant capacity (TAC) was also increased after 0.5 mM treatment which coincided with increased GR activity and elevated glutathione content indicating this low molecular weight antioxidant as an important factor associated with nickel tolerance. This study also emphasizes the possible important role of luminol-converting peroxidases in nickel hyperaccumulation, although they are not considered as antioxidant enzymes sensu stricto since some of them can also produce reactive oxygen species as well.

Intact and wounded shoots of in vitro cultured potato were investigated in situ to determine how their phototropic (PT) bending capacity was affected by water availability and exogenous auxin supplementation. Wounding strongly decreased PT bending but it recovered upon supplementation of water, auxin or both. Intact shoots required neither water nor auxin, while shoot segments required both. Shoot cuttings required only water, while, like shoot segments, decapitated shoots had a strong requirement for auxin. Water supplementation was beneficial in all treatments and PT bending was not affected in cultures that were submerged in water for a short period. Sucrose and inorganic salts present in the medium strongly affected PT bending capacity of cultures, favouring combinations with lower concentrations of both. Sucrose alone strongly promoted PT bending up to a concentration of 5%. Osmotic shock induced by the addition of small volumes of highly concentrated carbohydrate solutions (sucrose, glucose or sorbitol) induced a rapid but transient decline in PT bending capacity. These results indicate that water availability is a major factor that affects PT bending in potato plantlets.