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.

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.

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.

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.

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.

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.

The effect of abiotic (salicylic acid, jasmonic acid and methyl jasmonate) and biotic (chitosan and yeast extract) elicitors on the growth and xanthone accumulation in two hairy root clones of Gentiana dinarica Beck. was studied. The obtained results showed that clone 3 was more responsive to elicitor treatment than clone D. The production of dominant xanthone norswertianin-1-O-primeveroside was not significantly affected by either of the abiotic elicitor tested but was stimulated with chitosan treatment. The highest concentrations of all elicitors strongly increased the content of xanthone aglycone norswertianin, but simultaneously reducing the production of its glycoside norswertianin-1-O-primeveroside. The most efficient in enhancing norswertianin production was a 7-day treatment with salicylic acid (200 μM) and chitosan (50 mg l− 1), which yielded a 7.7- and a 24-fold increase in norswertianin content, respectively. In addition, treatment with biotic elicitors caused the occurrence of new xanthone compounds that were not detected in other samples. Free radical scavenging activity of xanthones was carried out by DPPH assay, and norswertianin showed the strongest activity.

The aim of the present study was to investigate the effect of sucrose on shoot regeneration potential in Hypericum perforatum L. roots obtained by Agrobacterium rhizogenes transformation. The morphological evaluation of transgenic roots grown on media supplemented with sucrose (0.5, 1, 2, 4, 6 and 8 %) indicated that both genotype and sucrose concentration significantly affected root elongation and branching, as well as shoot regeneration. For two of five analyzed clones, lower sucrose concentrations (up to 2 %) led to intensive shoot regeneration, while the other three clones intensified shoot development only at elevated sucrose concentrations (4 %). For all clones, concentrations above 4 % had a deleterious effect on both root and shoot development. Genetic characterization of regenerated shoots revealed that all tested clones were diploid with an average of 0.670 ± 0.002 pg of DNA per nucleus, with no significant differences between transgenic and non-transformed plants and, according to PCR, with integrated A. rhizogenes rolA, -B, -C and -D genes. Real-time RT-PCR confirmed the expression of rolA, -B and -C, while expression of the rolD gene was not detected. Differences were detected in the absolute amounts of transcripts between analyzed clones, with the highest levels of expression for all three analyzed rol genes in a clone previously defined as having high root differentiation and less effective shoot regeneration potential. The observed variations in morphogenesis potential could be attributed to different levels of expression of integrated rolA, -B and -C genes; while sucrose additionally pointed out these trends.

Phototropic (PT) and gravitropic (GT) bending are the two major tropic movements that determine the spatial position of potato shoots. We studied PT bending of potato plantlets grown under long-day photoperiods in several prearranged position setups providing different interactions with the GT response. Starting with the standard PT stimulation setup composed of unilateral irradiation of vertically positioned shoots, experiments were also done in antagonistic and synergistic setups and in treatments with horizontal displacement of the light source. In the standard setup, PT bending suppressed the GT bending, which could occur only if the PT stimulation was cancelled. The antagonistic position, with phototropism and gravitropism attempting to bend shoots in opposite directions, showed phototropism and gravitropism as independent bending events with the outcome varying throughout the day reflecting diurnal changes in the competence of individual tropic components. Whilst gravitropism was constant, phototropism had a marked daily fluctuation of its magnitude with a prominent morning maximum starting an hour after the dawn in the growth room and lasting for the next 6 h. When phototropism and gravitropism were aligned in a synergistic position, stimulating shoot bending in the same direction, there was little quantitative addition of their individual effects. The long period of morning PT bending maximum enabled multiple PT bending events to be conducted in succession, each one preceded by a separate lag phase. Studies of secondary PT events showed that potato plantlets can follow and adjust their shoot position in response to both vertical and horizontal movements of a light source. PT bending was reversible, since the 180° horizontal change of a blue light (BL) source position resulted in reversal of bending direction after a 20-min-long lag phase.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.

Study describes protocols for in vitro propagation of Gentiana utriculosa L. via axillary shoot multiplication and indirect somatic embryogenesis. Shoot cultures were established from seedling epicotyl explants cultured on MS medium supplemented with 0.25 mg L−1 BA and 0.1 mg L-1 IAA. Medium containing 2% sucrose and 0.2 mg L−1 BA improved multiple shoot production, providing 2.3 shoots per explant. The highest rooting (29.6%) was obtained on medium with 1/2 MS mineral salts and 0.5 mg L−1 NAA. Somatic embryogenesis was induced using different explants, including immature seeds as well as leaves and roots from shoot cultures. Following auxin treatment with either 1.0 mg L−1 2,4-D (immature seeds and leaves) or 0.1 mg L−1 NAA (roots), explants produced embryogenic calli which upon transfer to plant growth regulator-free medium allowed embryo conversion into plantlets. The best embryogenic response (82%) was obtained in calli derived from leaves cultured with their abaxial surface in contact with medium, whereas the highest embryo conversion rate (68%) was recorded for calli induced on immature seed explants. Histological analysis in all explant types revealed development of proembryogenic cell complexes at callus periphery, giving rise to somatic embryos. The presence of embryos at various stages of development indicated asynchronous somatic embryogenesis in G. utriculosa. Derooted embryo-derived plantlets placed on medium with 0.2 mg L−1 BA multiplied further as shoot cultures.

Kohlrabi (Brassica oleracea var. gongylodes) cultivars Vienna Purple (VP) and Vienna White (VW) were tested for their ability of de novo organogenesis in vitro. Root, cotyledon, hypocotyl explants and intact seedlings were cultivated on Murashige and Skoog (MS) media supplemented with different cytokinins: benzyladenine (BA), thidiazuron (TDZ), trans- or cis-zeatin. All tested cytokinins, including cis-zeatin, induced shoot regeneration from hypocotyl explants and intact seedlings, with seedlings being most successful for regeneration efficiency and viability of regenerated shoots in both cultivars. The highest frequency of shoot regeneration was achieved on MS with BA (60 %) or TDZ (50 %) for VP; and with BA (50 %), TDZ (47.5 %) or transZ (37.5 %) for VW. Measurements of the endogenous cytokinin and indole-3-acetic acid (IAA) contents in both hypocotyl explants and seedlings with regenerated shoots (HRSs and SRSs) suggested that the observed differences in organogenic response between these two types of explants were related to their cytokinin and IAA contents. HRSs generally exhibited elevated amounts of total cytokinins, while SRSs displayed a higher IAA/bioactive cytokinins ratio. Shoots regenerated from seedlings were further successfully multiplicated on a medium supplemented with BA (0.5 mg L−1). The rooting potential of multiplicated shoots was tested on media supplemented with 2 or 4 mg L−1 indole-3-butyric acid (IBA), with the higher concentration of IBA leading to more efficient rooting. Rooted plantlets were successfully planted into soil and flow cytometric analysis did not reveal ploidy variations, indicating that the described protocol is fast and efficient for kohlrabi regeneration.

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Strain 15834/PI had higher induction rate of hairy roots (32.15 %) than strain A4M70GUS (6.12 %). Transgenic nature of the roots was confirmed by GUS assay and PCR analysis. Two clones per strain (A4M70GUS-D and -I, and 15834/PI-2 and -3) with marked differences in general morphology and growth rate were further studied. The methanol extracts of hairy root clones were analyzed for xanthones content using HPLC method. The effects of the type of carbohydrate source (sucrose, fructose or glucose) at different concentrations on the growth parameters (growth index, dry weight, fresh/dry weight ratio), phenolic and xanthone production, and free radical scavenging activity of the transgenic clones were evaluated. Statistical two level factorial design was used to define optimal conditions for growth and successful secondary metabolite production in G. dinarica hairy root clones. The results showed that clones A4M70GUS-D and 15834/PI-3 were the superior ones. These two clones had the highest dry weight on 116.8 mM sucrose, producing up to threefold higher amounts of total phenolics and norswertianin-1-O-primeveroside than other clones, untransformed roots and roots of wild-grown plants.

Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato.

The phototropic (PT) bending capacity of potato plantlets in vitro was studied under conditions of continuous light (CL) and subsequent darkness (CLD). In CL, diurnal changes characteristic for the 16/8 h LD photoperiod were absent and the response, although complex, was constant at any time of the day. Graphic representation of shoot bending indicated the presence of two separate, partly overlapping PT responses. The first was a fast, time-limited process (FPR) with a 20 min lag phase and a maximum fixed at 70 min, followed by a steady bending process (SPR) starting 50–90 min after the beginning of blue light (BL) stimulation. The FPR response rapidly deteriorated in darkness and after 60–120 min it was replaced with BL augmented circumnutations. SPR was a constant feature in darkness and while both FPR and circumnutations were absent when darkness exceeded 120 min, the SPR response remained present in darkness irrespective of its duration. The abrupt end of circumnutations marked the beginning of the synchronized SPR bending response. Darkness lasting 120 min was also a pivotal point after which the PT capacity started to recover. After 8 h of CLD, the PT capacity was fully restored. The FPR response reappeared at dawn when the light was turned on. Potato plantlets also manifested fast solar tracking movements, correcting shoot position in relation to the horizontal displacement of the BL source. Solar tracking movements contrary to circumnutations comprised shoot torsion twisting (twining). Our results demonstrate that, in potato shoots, multiple PT responses are present and operational simultaneously.