Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase

Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Gal1,3(Fuc1,4)GlcNAc, on some N-glycans. By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian 1,3/4-fucosyltransferases. The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (K_m 16 M) to lacto-N-tetraose (Gal1,3GlcNAc1,3Gal1,4Glc; K_m 80 M) as well as to 1,3- and 1,4-galactosylated N-glycans. It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Plant regeneration from hypocotyl segments of Lupinus luteus cv. L. Aurea

Complete plants of Lupinus luteus L. cv. Aurea that were regenerated from hypocotyl segments, bloomed, produced seeds and were efficiently nodulated by Bradyrhizobium sp. strains. The highest rates of shoot formation were obtained on A medium plus 1.3% agar with 10.0 M 2-isopentenyladenine (2iP) and 0.11 M naphthaleneacetic acid (NAA); the best rooting was achieved on a medium with 0.5 M NAA plus 0.05 M 2iP. Afterward, plantlets were transferred to either perlite or peat-containing pots and irrigated with a N-free nutrient solution until maturity. Direct rooting of hypocotyls could also be obtained on A medium with 1% agar.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzylaminopurine - GUS -glucuronidase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - EtOH ethanol - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - NOS nopaline synthase - CaMV cauliflower mosaic virus     

Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 g), lipopolysaccharide (0.1–100 g/ml), tumor necrosis factor- (TNF) (3.13–50 ng/ml), or interleukin-1 (IL-1) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF reduced the mitotic index by 45%, slowed cell cycle progression by 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1 interleukin-1 - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNF tumor necrosis factor      

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

Fine structure of abscission zones

Summary Electron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 m in width while the main groove averages approximately 200 m in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 m in thickness and are spaced at 4 m intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 m and 1,500 m in profile. In tomato they average 300 m and 500 m. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 in diameter in chloroplasts ranging from 1.6 to 2.2 in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 m are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

Improved plant regeneration from barley callus cultures by increased copper levels

Incorporation of cupric sulfate into callus induction, maintenance, and regeneration media significantly enhanced plant regeneration from callus cultures of barley (Hordeum vulgare L.) immature embryos. Embryos from the cultivars Hector and Excel were cultured on MS medium containing 0, 0.1 (MS level), 0.5, 1.0, 5.0, 10.0, 50.0, or 100.0 M cupric sulfate. Plants were regenerated beginning at 8 weeks and continuing through 36 weeks. For Hector, medium containing 50 M copper regenerated significantly more plants than any other medium, with an average of 17 plants per embryo. In comparison, medium with MS copper levels (0.1 M) regenerated only 5 plants per embryo. For Excel, medium containing 5.0 M copper was the best, regenerating 1.4 plants per embryo. No Excel regenerants were obtained on medium with MS copper levels. Increased copper levels also increased the percentage of embryos that regenerated at least one plant, in both cultivars. The results indicate that MS copper levels are not optimized for barley callus cultures, and that improved plant regeneration can be obtained at higher copper concentrations.Abbreviations MS Murashige & Skoog (1962) - 2,4-d 2,4-dichlorophenoxyacetic acid The US Government's right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged