Measurement of endogenous ABA levels in chilled somatic embryos of carrot by immunoassay

Somatic embryos of carrot, Daucus carota L. Royal Chantenay, were chilled at 4°C for the last 3 days of development in order to harden torpedo stage embryos to increase embryo survival during desiccation. ABA levels in chilled and non-chilled embryos were measured using a polyclonal radioimmunoassay and a monoclonal enzyme-linked immunosorbent assay (ELISA). The monoclonal ELISA is the preferred technique due to superior sensitivity and specificity. ABA levels, measured by either technique, were similar in chilled and non-chilled embryos. The relative water content was lower in chilled embryos than in non-chilled embryos and chilling altered protein secretion of one cell line.List of abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BHT 2,5-di-tert-butyl-4-methyl phenol - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay.     

Estimation of fungal growth on lignocellulosic substrates using an enzyme-linked immunosorbent assay

Summary The feasibility of using an enzyme-linked immunosorbent assay (ELISA) for quantifying the growth ofThielavia terrestris on cellulosic substrates was studied. WhenT. terrestris was grown, using glucose as the substrate, the ELISA readings correlated well with the mycelial dry weight values. When lignocellulosic substrates were used, the ELISA appeared to slightly overestimate fungal growth. The ELISA values indicated that the fungi grew more slowly on steam-treated aspenwood than on commercial sources of cellulose (Avicel).     

Isolation,culture and callus regeneration of protoplasts of wild and cultivated Helianthus species

Protoplasts were isolated from seedling roots, hypocotyls, and cotyledons of four cultivars of Helianthus annuus and from leaves of axenic shoot cultures of the wild species H. praecox, H. scaberimus and H. rigidus. Optimal culture conditions were established for the respective protoplast systems, using the agarose bead method of culture. Protoplast division was induced for all the species examined. In the case of the cultivars of H. annuus, hypocotyl and cotyledon protoplast division was sustained leading to callus formation, which in turn, could be induced to produce roots and organised meristematic regions in the presence of NAA and 6-BAP.Abbreviations 6-BAP 6-benzylaminopurine - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

Regeneration of plants from achenes and petals of Chrysanthemum coccineum

Achenes and petals of Chrysanthemum coccineum were cultured on MS and White medium supplemented with BA and NAA or 2,4-D. Without being transferred, shoots were formed directly and immediately after callus formation on the surface of the achene walls and from the cut ends of the petals. High concentracions of BA and NAA supported the callus induction and shoot formation, but higher concentrations of 2,4-D inhibited shoot formation. The shoots obtained from both explants formed roots when transferred to hormone-free medium and they could be transplanted to soil for further growth. The regenerated plants contained as much pyrethrins as the original plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthalene acetic acid - BA 6-benzyladenine     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Tissue culture of Chrysosplenium americanum and its potential for flavonoid production

Chrysosplenium americanum (Saxifragaceae) accumulates a variety of partially O-methylated flavonol glucosides. Because of the semi-aquatic nature of this plant and its extensive contamination with endogenous organisms, the initiation of shoot and callus cultures could only be achieved after (a) using a special surface sterilization procedure, (b) production of new shoots from initial explants, and (c) selective elimination of organogenic structures during several subcultures. HPLC analysis of the cultured tissues established the presence of a number of flavonoids characteristic of the intact plant, though in lower and variable concentrations. However, shoot and callus cultures exhibited flavonoid profiles similar to those of the intact leaves and roots, respectively.Abbreviations BAP benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - HPLC high pressure liquid chromatography     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

Determination of endogenous abscisic acid levels in immature cereal embryos during in vitro culture

Levels of endogenous abscisic acid (ABA) in immature wheat (Triticum aestivum cv. Timmo) and barley (Hordeum vulgare cv. Golden Promise) embryos have been determined by enzyme-linked immunosorbent assay. Embryos of both cereal species showed an increase in ABA content during development on the parent plant. Immature embryos were excised and cultured in vitro on nutrient media that led to precocious germination or on media containing 9% (w/v) mannitol that maintained their developmental arrest. Barley and wheat embryos responded to these culture conditions in an identical manner with respect to changes in morphology, fresh weight, protein and lectin content. However, in complete contrast, the ABA content of barley embryos increased by an order of magnitude during culture on mannitol, whereas that of wheat embryos showed no significant change. The results are discussed within the context of the role of ABA in the regulation of embryo development.Abbreviations ABA abscisic acid - BGA barley-germ agglutinin - dpa days post anthesis - ELISA enzyme-linked immunosorbent assay - GC-MS gas chromatography-mass spectrometry - WGA wheat-germ agglutinin     

Callus formation from Malus x domestica cv. ‘Jonathan’ protoplasts

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - IAA indole acetic acid     

Immunochemical quantification of cytochrome f in leaves of a non-yellowing senescence mutant of Festuca pratensis

A non-competitive enzyme-linked immunosorbent assay (ELISA) which enables detection of as little as 0.1 ng cytochrome f in leaf extracts has been developed. No evidence for specific or non-specific interference by proteins other than cytochrome f was found. The assay was applied to a comparative study of age-related changes in the cytochrome f content of leaves of Festuca pratensis Huds. cv. Rossa, and a non-yellowing mutant genotype (Bf993) having a lesion in the mechanism responsible for thylakoid membrane disassembly. Cytochrome f in senescent leaves of the latter genotype was found to be present at significantly higher levels than in the wild-type, implying an inability on the part of the mutant to degrade this protein. The results obtained by ELISA were confirmed by antibody probing of Western blots.Abbreviations Ab antibody - BSA bovine serum albumin - chl chlorophyll - cyt cytochrome - ELISA enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b binding protein - OEC oxygen-evolving complex - PAGE polyacrylamide gel electrophoresis - POase peroxidase - SDS sodium dodecyl sulphate - PS II Photosystem II - TBS Tris buffered saline - Tris tris (hydroxymethyl) aminomethane     

Translocation and multiplication ofSpiroplasma citri in turnip (Brassica rapa)

Following inoculation of designated leaves of turnip plants withSpiroplasma citri byCirculifer tenellus, spiroplasmas were cultured first from roots (four days) and then from youngest leaves (eight days), but almost never from oldest leaves. In experiments using enzyme-linked immunosorbent assay to monitor changes in titer in turnip leaves during the course of plant infection,S. citri was detected seven days after inoculation and reached peak titers of 10¹⁰–10¹¹ colony-forming units/g 12–20 days after inoculation, declining thereafter. Spiroplasmas were detected 5–9 days before symptoms appeared.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Participation of an active transport system in berberine-secreting cultured cells of Thalictrum minus

The release of the benzylisoquinoline alkaloid berberine from cultured cells of Thalictrum minus into the medium proved to be temperature-dependent and was suppressed by such inhibitors of the plasma membrane-bound ATPase as vanadate and diethylstilbestrol. These results indicate that berberine is secreted through an energy-requiring process located in the plasma membrane of berberine-producing T. minus cells. This is the first finding that a secondary metabolite of plant cell culture is secreted by an active transport system.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DES diethylstilbestrol - DMSO dimethyl sulfoxide - LS Linsmaier and Skoog (1965) - NAA 1-naphthaleneacetic acid     

Detection of beet yellows virus in sugar beet leaves and roots by enzyme-linked immunosorbent assay (ELISA)

Using the enzyme-linked immunosorbent assay (ELISA) beet yellows virus (BYV) could be detected reliably in the leaves of sugar beet andTetragonia expansa Pall. and in the roots of sugar beet. Specifio γ-globulin of BYV antiserum was coupled to horse radish peroxidase by periodate oxidation. Optimum dilutions of antigen (extract from infected leaves) were1: 50 to 1: 200 for BYV detection in sugar beet andT. expansa leaves and1: 2 to 1: 5 for detection in sugar beet roots. Extracts from beet roots are not to be purified by ultracentrifugation, however, by the described method virus can be demonstrated only in 80–90% of naturally infected sugar beet roots. The method is specific, no increase of extinction values was found in healthy or beet western yellows virus infected plants. Presence of virus can be demonstrated by visual as well as photometric evaluation. Results confirmed the suitability of peroxidase application for detection of plant viruses by ELISA.     

Effect of cytokinin on morphological changes of suspension cultured cells of the moss, Barbula unguiculata

Suspension cultured cells of the moss, Barbula unguiculata, grow actively in both light and dark culture. Light-grown cells contain chlorophyll and exhibit an undifferentiated callus form. When cells are transferred to a dark condition, they develop into protonemata. Protonemata formation in the dark can be inhibited by the addition of 5 M benzyladenine or 6-furfurylaminopurine but is not affected by the addition of 5 M 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - kinetin 6-furfurylaminopurine - NAA naphthalene acetic acid     

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy roots synthesized the same saponins, ginsenosides, as those of the native root, up to about 2.4 times in the quantity, and up to about 2 times in comparison with that of the ordinary cultured roots, on dry weight basis.Abbreviations Ms medium Murashige and Skoog's medium - 2,4-D 2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - K kinetin This paper is Part 52 in the series "Studies on Plant Tissue Culture". For Part 51, see Ayabe S, Udagawa A, Furuya T (1987).     

Production of granulocyte colony-stimulating factor by head and neck carcinomas

Detectable levels of G-CSF by enzyme-linked immunosorbent assay (ELISA) were found in sera of 4 out of 15 patients with head and neck carcinomas. Also cells prepared from the tumors of these 4 patients secreted G-CSF. The supernatants of cells derived from all 15 patients did not contain granulocyte-monocyte CSF, monocyte CSF, tumor necrosis factor-, transforming growth factor- ₁, epidermal growth factor, interleukin (IL)-1 and IL-6. These findings suggest that leukocytosis in patients with carcinomas might be due to the production of G-CSF by tumor cells.Abbreviations CSF colony stimulating factor - EGF epidermal growth factor - ELISA Enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IL interluekin - M monocyte - TGF transforming growth factor - TNF tumor necrosis factor