Plant Callus Tissue Research Articles

Elucidating the Metabolism of 2,4-Dibromophenol in Plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized inthe plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

Allergy-Protective Arabinogalactan Modulates Human Dendritic Cells via C-Type Lectins and Inhibition of NF-κB.

Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3 -: grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif -: containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs.

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

SummaryFungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.

Antimicrobial activity of flavonoids from in vitro tissue culture and plant parts of two plant species of Western Rajasthan

Extract of flavonoids from plant parts and callus tissues of Glinus lotoides (molluginaceae) and Psoralea odorata (fabaceae) were screened against microbes’ viz., Bacillus cerus, Escherchia coli, Salmonella typhimurium, Staphylococcus aureus and Candida albicans adopting disc diffusion method. Results were compared with the zone of inhibition produced by commercially available standard antibiotics. Maximum activity was observed in flavonoid of callus tissue as compared to plant parts of both plant species.

Production of phytohormones, siderophores and population fluctuation of two root-promoting rhizobacteria in Eucalyptus globulus cuttings

Vegetative propagation by stem cuttings and mini-cuttings has been used worldwide for growing Eucalyptus plants. However, clones and hybrids of this plant present a great variability in their rooting capacity, apart from a gradual decrease in the rooting potential due to the ontogenetic age of the mother plant. Several studies have demonstrated that some bacteria promote plant growth and rooting through the action of direct and indirect mechanisms that are not still completely clear. Considering this, the objective of this study was to assess the production of auxins, abscisic acid and siderophores in Bacillus subtilis and Stenotrophomona maltophilia, which in previous studies increased rooting of E. globulus cuttings. Additionally, the population of these bacteria in the rhizosphere, superficial tissues of the stem-base and callus of the mini-cuttings was identified, and quantified by real-time PCR. Only S. maltophilia produced IAA in the presence of tryptophan; none of the bacterial strains produced ABA, but both produced siderophores. A comparative analysis of the separation profiles showed that there is a diverse microbial community in the rhizosphere, and only S. maltophilia was capable of keeping its population at a density of 2.03 × 10(7) cells/mg in different tissues of the mini-cuttings. The results would indicate that the rooting stimulus in E. globulus could be related to the action of one or several mechanisms such as the production of auxins and siderophores, and it could also be associated with the ability of bacteria to stay in the rhizosphere or in plant callus tissues.

Propagation and Effects of the Pinewood Nematode on Calli of Various Plants

Bursaphelenchus xylophilus and its closely related species, B. mucronatus were successfully propagated well on calli of tobacco (Nicotiana tabacum), alfalfa (Medicago sativa) and several pine species. Different propagation rates or different maximum populations were recorded. Most nematodes occurred in the outer layer of the callus, in which cells appeared to be dead. Tannin accumulated in the nematode- inoculated callus tissues. The correlation of the tannin content with the number of Many studies have been done on pine wilt disease caused by the pinewood nematode, Bursaphelenchus xylophilus (STEINER and BUHRER) NICKLE. Several of these studies have focused on host resistance and on chemical components of the host that attract nematodes or promote their reproduction3' 15) as well as factors that inhibit invasion of host tissue3) or affect nematode mobilityn. FUTAI and FURUNO7 who inoculated the pinewood nematodes into young trees of 30 pine species, showed that resistance to the pinewood nematode varies among pine species. They correlated the differences in host resistance to taxonomic categories of pines (sensu CLITCHFIELD and LITTLE, 1966), but could not explain the cause of such differences. The mechanisms of pine wilt and host resistance have been studied by propagating B. xylophilus on tissue cultures of various plants. For example, TAMURA and MAMIYA13, 14) observed that B. xylophilus feeds and reproduces on the calli of Pinus densiflora and P. thunbergii, both of which are susceptible to pine wilt. TAMURA and DROPKINI1) propagated B. xylophilus on the callus of P. jeffreyi which is a resistant species to pine wilt. B. xylophilus has been propagated on calli of non-host plants such as alfalfa (Medicago sativa)12) and Japanese red cedar (Cryptomeria japonica). It is also well-known that B. xylophilus can be propagated on various fungi. These examples demonstrate the nematode's ability to feed and reproduce on various kinds of plant callus tissues and fungal cells. In the present study, the population buildup of B. xylophilus and that of non-pathogenic related species B. mucronatus were compared on calli of four pine species and alfalfa. In addition, the reproduction of B. xylophilus was also examined on calli of the F1 hybrid of P. thunbergii x P. massoniana and N. tabacum. Special consideration was also paid to the effect of nematode feeding and population buildup on callus responses, and to the way this relates to resistance shown in nursery trees. Tannin production in inoculated callus was also determined, which is known to be associated with host resistance to the pinewood nematode6). Nematode feeding and movement on callus cultures was also observed microscopically.

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.

Infection of plants and plant tissue cultures with cyanobacteria-bacteria complexes

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria–bacteria associative microsymbiont complexes (AMC) isolated from natural syncyanoses (the ferns Azolla pinnataand Azollasp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with AMC led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the AMC isolated from the fernsA. pinnataand Azollasp., the callus tissue specifically influenced the growth of the AMC components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the AMC components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.

Interaction of ericoid mycorrhizal fungi with host and non host plant callus tissues

Interaction of ericoid mycorrhizal fungi with host and non host plant callus tissues

Polarographic investigation of some cytokinins

The cytokinins constitute one of the five groups of plant hormones, and are characterised by their ability to promote cell division in cultured plant callus tissue under appropriate conditions [I-31. With the exception of diphenylurea, all cytokinins that have been unambiguously identified in extracts of higher plants are derivatives of adenine, including 6-aminoand riboside-substituted compounds. While the electrochemical behaviour of other adenine derivatives has been explored quite extensively [4-121, no study has yet been reported on this important class of biologically important adenine derivatives. We have, therefore, carried out a preliminary investigation into the polarographic behaviour of the following cytokinin derivatives as a prelude to more detailed investigations in this topical field of research.

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Callus tissue ofHaworthia mirabilis Haw. was irradiated with60Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N6-(Δ2-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N6-(Δ2-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N6-(Δ2-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed.

In vitro neoplastic transformation of plant callus tissue by γ-radiation

Tumors have been induced by γ-radiation in callus tissue derived from a monocotyledonous flowering plant, Haworthia mirabilis Haw. The transformed tissue exhibited compact texture, excessive cell proliferation and loss of capacity for organogenesis. Tumors were characterized by their ability to undergo continuous autonomous growth on minimal media in the subsequent 4 generations of subculture. In contrast, the nonirradiated control tissue grew with friable texture, required inositol or growth hormones and showed prolific differentiation of vegetative buds.

Metabolism of 2,4-dichlorophenoxyacetic acid. VII. Comparison of metabolities from five species of plant tissue cultures.

Metabolism of 2,4-dichlorophenoxyacetic acid. VII. Comparison of metabolities from five species of plant tissue cultures.

Organogenesis in isolated carnation plant callus tissue cultivatedin vitro

Formation of stems both in callus tissue isolated from hypocotyl and in apical meristems culture of carnation plants (Dianthus caryophyllus L. ev. “Grenadin white, yellow, scarlet red, dark red and pink”) was evokedin vitro using chemically defined medium. The rooted stems were transferred into pots and cultivated under natural conditions.

3-Methylpurpurin and other anthraquinones from callus tissue of Digitalis lanata

Four additional anthraquinones, one of them new, have been isolated from the callus tissue of Digitalis lanata, from which 4-hydroxydigitolutein (V) and digitolutein (VI) have previously been obtained. The new pigment is 3-methylpurpurin (III) and the others are 3-methylquinizarin (I), pachybasin (II) and 3-methylalizarin (IV). This is the first report of I, II, III and IV as metabolic products of plant callus tissues.

Propagation of Telotylenchus loofi on plant callus tissues.

Attempts have been made to cultivate Telotylenchus loofi on plant callus tissues at different temperature ranges. Although the subjection of cultures to 17–20, 30–35 and 35–40° C temperatures did not prove a handicap in nematode propagation, huge populations could only be obtained at 25–27° C. The temperatures below 17° C and beyond 40° C were found unsuitable for cultivation of T. loofi.

Growth and chlorophyll production in plant callus tissues grown in vitro

Growth, nutrition and chlorophyll development were studied in chlorophyllous callus tissues isolated from the following edible angiospermous plants: carrot root, crown gall of tomato, endive embryo, leaf petiole and stem of lettuce, leaf petiole of parsley, pea stem and rose stem. Growth patterns of these tissues in vitro were sigmoid. Synthetic media produced less growth, in terms of fresh weight increase, than media containing coconut milk, a highly complex and little understood natural substance. MURASHIGE and SKOOG'S synthetic medium proved useful for satisfactory growth and chlorophyll production in a number of tissues. Its usefulness was further increased by additional amounts of copper sulphate, potassium nitrate and monobasic ammonium phosphate. Increased levels of iron and magnesium inhibited growth. Incorporation of yeast extract in the tobacco-high-salts-medium produced the highest amount of growth and chlorophyll formation in endive tissue. Presence of exogenous sucrose was essential for the continued good growth of the above callus tissues in vitro. Highest amount of growth took place either in white light or in the dark. Different tissues had different responses to high or low intensities of light. Endive and carrot tissues produced in vitro were palatable to human taste. Endive tissue was particularly good as it also differentiated many small rosettes of leaves, shoots and had a mild aromatic flavor typical of the endive plants grown in nature.

Transmission of Tobacco Necrosis Virus to Tobacco Callus Tissues by Zoospores of Olpidium brassicae

MOREL1 infected cultures of plant callus tissue with some, but not all, of the obligate parasites he tested. Thus, with fungi, he succeeded with Plasmopara viticola and Uncinula necator, but not with several species of rust fungi on callus from susceptible host plants. He also infected tobacco callus with tobacco mosaic virus, but this is difficult and most work with plant viruses in tissue cultures has consequently been done with tissues taken from plants already systemically infected. Unwounded callus tissues are immune from infection by viruses, and although tissues can be infected by pricking with fine needles dipped in concentrated inoculum, the method is not always successful2. We have now found that tobacco callus tissue can be readily infected with tobacco necrosis virus with the aid of Olpidium brassicae, an obligately parasitic fungus that was known previously only to infect plant roots. Teakle3,4 showed that Olpidium aids the infection of roots by this virus, and we have confirmed this.

FERN CALLUS TISSUE CULTURE

THE FIRST INDEFINITE plant callus tissue cultures were obtained by White (1939), Gautheret (1939), and Nobecourt (1939), from tumorous tissue or from fleshy organs composed for the most part of undifferentiated parenchyma. Subsequently it proved feasible to cultivate tissues obtained by the proliferation in vitro of cambium and phloem parenchyma of various herbaceous and woody dicotyledons, and more recently it has been found possible to obtain tissue cultures from monocotyledons (Morel and Wetmore, 1950). Vascular cryptogams, and specifically ferns, on general grounds would not seem to be very favorable for tissue culture studies. They are, in fact, devoid of secondary activity and are almost entirely made up of highly differentiated tissues seemingly unlikely to proliferate masses of meristematic parenchyma, which tissue is conducive to growth in vitro. General observations, however, led to the belief that this problem was not impossible to solve. For example, in the course of his investigations on regeneration in ferns, Goebel (1907) and his students (Beyerle, 1932) observed several times growth of undifferentiated formations on the petiole and blade of sporeling leaves which he had removed from the plant and maintained on damp soil. These outgrowths were analogous to callus of dicotyledons. The early observations of Farlow (1874), de Bary (1878), Heim (1846), and others more recently showed that a similar type of formation may also appear on the prothalli of certain species of ferns. However, in these cases the proliferation was usually followed by the appearance of apogamous sporelings. It was such proliferations as these that we employed to obtain callus cultures of fern. Spores of Osmunda cinnamomea L. were planted in March 1948, on a medium devised by Knudson (1925) in the course of his investigations on orchid embryo cultures. They germinated in a few days and developed rapidly forming thin heart-shaped prothalli. After 2 months of culture, we observed on five of these prothalli the spontaneous appearance of undifferentiated calluses. These calluses appeared on the upper surface and were formed of a mass of greenish cells containing fewer chloroplastids than those of the remainder of the prothallus. They developed slowly and by June 1949, were transferred to the same medium, upon which their growth continued very slowly. After 8 months, the largest had attained a diameter of only 1 cm., while