Influence of humus substances on the toxic effect of 2-methyl-4-chlorphenoxyacetic acid

Effect of humus fractions on the toxicity of 2-methyl-4-chlorphenoxyacetic acid (MCPA) was examined in water cultures. Humus fractions and different MCPA quantities were added to Richter's nutrient solution used for maize (Zea mays L., cv. Ko?ovská raná) cultivation. The results show that MCPA and humus substances in the applied concentrations intensively influence the maize root system, especially the rhizodermis and apical meristem cells. MCPA reduces ion absorption, induces phosphorus excretion from root, reduces tissue hydratation and relatively quickly kills these cells, at the end stage and/or in the highest concentration. Humus fractions have an unambiguously positive effect on these processes, they increase the cell vitality. When humus substances are applied, the MCPA effect does not appear in potassium and nitrogen absorption, the inhibitory effect is maintained in phosphorus absorption and in tissue hydratation even with the most effective fraction—with fulvo acids. But all fractions increased the cell vitality of sorption tissue and meristems in the presence of MCPA.     

Development of embryoids in carrot root callus culture (Daucus carota L.)

Embryogenesis occurred in carrot root callus (Daucus carota L.) cultivated on simple synthetic medium containing IAA and 2,4-D. Embryoid development continued also during successive years when the tissue was cultivated on the same nutrient medium without those substances. Sometimes production of plants with atypical leaves was also observed. In those plants development of adventive embryoids occurred repeatedly. Result of this work confirmed reports about the organogenic potentiality of this species and about its sensitivity to some chemical substances.     

In vitro induction of cell division in the epidermis of stem segments ofTorenia fournieri Lind.

Cell division in the epidermis of stem segments ofT. fournieri stopped immediately when the epidermis was separated from subjacent tissues after having been in contact with these tissues for some time. Thus, the effects of the inductive signals emanating from these tissues did not persist. However, cell division in isolated epidermis cultured alone could be induced by adding asparagine, alanine or glutamine to the medium. Asparagine, at 5 mM, had the greatest stimulatory effect. Growth substances had a synergistic effect on this induction by amino compounds. However, these cell divisions, unlike those in epidermis cultured together with subepidermal tissues, did not lead to organogenesis. The amino compounds which partially replaced the inductive action of subepidermal layers on the epidermis can be considered as one of the endogenous factors coming from the first-named layers in intact explants.     

Elimination of mycoplasma in tobacco callus tissues (Nicotiana glauca Grah.) culturedin vitro in the presence of 2,4-D in nutrient medium

Callus tissue cultures were established from stems of tobacco plants (N. glauca Grah.) both healthy and mycoplasma (potato witches' broom disease) infected on a modified nutrient medium (with a lower content of mineral salts) according toMurashige andSkoog (1962) in the presence of 2,4-D (1 mg l^?1) as a growth regulator. No differences were observed in the growth and development of both tissues. Organogenesis appeared on a nutrient medium (Petr? et al. 1972) supplemented with kinetin (0.64 mg or 2.56 mg l^?1) and IAA (2 or 4 mg l^?1). Callus derived from mycoplasma diseased plants started to form numerous buds after three months whereas organogenesis in callus from healthy controls appeared only after six months. We suppose that the reason of this difference is the fact that an expressively higher content of 2,4-D was found in the calli from healthy plants in comparison with the corresponding tissue from mycoplasma diseased ones. Reconstituted plants were isolated, rooted and transferred in the soil. The infectivity of these plants was assayed by grafting their stem tips on tomato plants which indicate very reliably and sensitively this mycoplasma disease. 31 reconstituted plants were obtained in the whole from calli isolated from mycoplasma infected plants and all of them were healthy. It was established that mycoplasma failed in the presence of 2,4-Din vitro. Stem pieces from diseased plants in which mycoplasma presence was proved, lose their infectivity after 4 weeks of cultivation on nutrient medium with this growth regulator. On the contrary 2,4-D which spreads and acts especially through phloem (Smith et al. 1947) does not kill mycoplasmain vivo even in doses evoking strong symptoms of 2,4-D effect on experimental plants.     

Growth and infectivity of callus cultures of tomato plants infected with a mycoplasma disease — Potato witches' broom

Callus tissues were derived from the stem of healthy tomato plants (Lycopersicum esculentum Mill. ev. Pr?honické) and of plants infected with potato witches' broom—a disease caused by mycoplasma. Callus cultures were established on modified fully synthetic media described byMorel (1948) and byMurashige andSkoog (1962). Callus cultures obtained from diseased plants were grown and subcultured on both media, growth in primary isolates from healthy plants took place on the Murashige and Skoog medium only. Growth of callus tissue derived from diseased plants was more vigorous even after several subcultivations in comparison with callus tissues isolated from healthy plants. Variations in the morphology in these callus cultures were not noted. Callus cells of diseased plants varied in size; they were about 50% larger than those from healthy ones. Implantation of primary and subcultivated callus tissues into tomato stems of healthy plants did not show any symptoms of infection on test plants.     

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l^?1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.     

Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientale and its relationship to alkaloid and lipid metabolism

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently induced somatic embryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In the P. somniferum cultures protopine and morphine type alkaloids (morphine, codeine, thebaine) appeared about 45 days after exchanging the medium. Thebaine was the main alkaloid in the P. somniferum embryoids accumulating up to 0.2 % of dry weight.     

Expression and effects of epidermal growth factor on human periodontal ligament cells

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.     

Physiological activities of ergoline alkaloids in submerged cultures ofClaviceps paspali andClaviceps purpurea

Ergoline alkaloids do not belong to a group of physio ogically inert secondary metabolites. Ergometrine as well as ergotamine increased the acetyl-CoA carboxylase activity and inhibited the activities of citrate synthase and isocitrate lyase. The slightly inhibitory effect of ergotamine and stimulatory effect of ergometrine on malate synthase was established. Supplied ergometrine brought about a change in the culture growth rate,i.e. inhibition (10^?4 m) or stimulation (10^?3 m). Addition of 10^?4 m ergometrine to the culture ofClaviceps paspali MG-6 increased the total alkaloid yield by 120%. The stimulatory influence was inversely proportional to the concentration of added alkaloid. It is assumed that accumulating alkaloids suppress attainment of the yield that would be achieved by continuous control of the alkaloid level. A positive relationship is assumed between the process of alkaloid synthesis and the viability of the culture.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

Preliminary studies of the effects of an induced phosphate deficiency on carbohydrate-nitrogen ratios inAlternanthera philoxeroides [Centrospermae: Amaranthacea] and on the feeding ofAgasicles hygrophila [Col.: Chrysomelidae]

In no-choice tests but not in choice tests, alligatorweed flea beetles,Agasicles hygrophila Selman & Vogt (Col.: Chrysomelidae) exposed to alligatorweed plants grown with two levels of mineral nutrition fed significantly more on those grown in full mineral nutrient than those grown with deficient phosphate. Chemical analysis showed that the full nutrient plants had more ethanol-soluble nitrogen compounds but less total carbohydrate than the phosphate-deficient plants. The response of beetles to phosphate-deficient alligatorweed may thus result from a change in the carbohydrate-nitrogen composition of host plants, though further investigation is needed for confirmation. Flea beetle response was identical to terminal and mature leaf tissue of full nutrient plants.     

Chromosomal instabilities in callus tissue from haploid barley (Hordeum vulgare L.)

Callus culture was derived from haploid barley embryos after crossing withHordeum bulbosum. The callus tissue is cytologically heterogeneous, containing haploid, diploid and polyploid cells. Aneuploidy and karyokinetic irregularities were also observed. Some problems of chromosomal instabilities in plant tissue cultures are discussed.     

The Down-Regulation of Neuroligin-2 and the Correlative Clinical Significance of Serum GABA Over-Expression in Hirschsprung’s Disease

The goal of this study was to investigate the expression level of neuroligin-2 in different colon tissue segments of children with Hirschsprung’s disease (HSCR) and the correlative clinical significance of serum Gamma-Aminobutyric Acid (serum GABA) in HSCR. Neuroligin-2 was assessed by Immunohistochemistry staining method on routine paraffin section from different colon tissue segments of HSCR (ganglionic colonic segment, transitional colonic segment and aganglionic colonic segment). Western-blot analysis and real-time fluorescence quantitative PCR(qRT-PCR) were applied to compare and evaluate the expression levels of neuroligin-2 from three segments of HSCR, and we used Enzyme-linked Immunosorbent Assay (ELISA) method to detect and compare the serum GABA between HSCR and non-HSCR. Immunohistochemistry staining demonstrated that intensive neuroligin-2 staining was detected in the ganglion cells in the ganglionic colonic and transitional colonic segments from the HSCR children; however, neuroligin-2 staining was down-regulated significantly in the aganglionic colonic segments. The expression levels of neuroligin-2 mRNA and protein in the aganglionic colonic segment were decreased compared to the ganglionic colonic segment and transitional colonic segment (P < 0.05). And the level of serum GABA was significantly higher in HSCR than that in non-HSCR. The expression of neuroligin-2 varies from different segments of HSCR. The down-regulation of neuroligin-2 in aganglionic colonic segments may be correlated with the excessive intestine contraction and further result in HSCR. The over-expression of serum GABA may be considered as a new diagnostic method of HSCR.     

Alkaloid synthesis is coupled to shoot morphogenesis in Argemone mexicana L. (Papaveraceae) in vitro cultures

During the induction process of an in vitro callus culture of Argemone mexicana L. (Papaveraceae), the levels of two benzylisoquinoline alkaloids known as berberine and sanguinarine displayed opposing trends. While the berberine levels steadily decreased from the initial explant stage up to the early proliferation of unorganized parenchymatous cell masses, the sanguinarine content increased. Once the callus culture was established, sanguinarine was the primary alkaloid present and berberine could no longer be detected. However, upon shoot regeneration, the berberine accumulation recovered, but sanguinarine was found in the newly formed leafy tissue. After root formation, sanguinarine was relocated to this organ, whereas berberine was evenly distributed between both tissues. Explants from stem internodes did not form callus, and berberine—plus sanguinarine—containing axillary shoots emerged from lateral buds in the induction medium. In contrast to callus-derived shoots, no root formation was observed. Therefore, alkaloid synthesis in A. mexicana in vitro cultures is related to the level of tissue organization in different ways, and while berberine accumulation seems to require the presence of differentiated organs, this is not the case for sanguinarine. Moreover, leafy parts of rootless shoots acquired the capacity to accumulate sanguinarine, which is usually absent in aerial tissues of mature plants. However, when these shoots were rooted, sanguinarine was mainly located in the newly formed roots, while berberine was detected in the shoots at similar levels found in the roots.

     

Bud breaking and adventitious root formation inPanicum maximum Jacq

When stem cuttings were put in water the dormancy of the bud was broken. No inhibitory substances could be found in the leaves and no effect of exogenous growth substances could be detected. Dormancy of buds in the present case seems to be the result of the mechanical resistance imposed by the leaf sheath upon the bud. Gibberellic acid was very effective in promoting root formation in the woody stem cutting ofPanicum maximum and the present results point to a direct effect on root initiation by gibberellic acid.     

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC–DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9′,10′) bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7′,8′) cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.     

The isolation of tissue culture ofPopulus alba L. ‘Pyramidalis’

Tissue culture was isolated from the stem ofPopulus alba L. ‘pyramidalis’. Callus formation was observed since November till March (1974),i.e. till the formation of calluses suitable for further subeultivation. The most vigorous growth was obtained with the callus culture cultivated on the nutrient medium of DIAZ-COLONet al. (1972) on which more than 11 g of fresh matter was produced after 30 d at the end of the first year of cultivation in darkness, with inoculum weight 1.5-1.8 g. A mild decrease in growth rate of the tissue culture was observed after the first year of cultivation. When illuminated, the originally yellow calluses turned green. The morphological and anatomical structure of the callus culture is also described and cell shape and cell size evaluated.