Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Tissue-specific activation of the osmotin gene by ABA,C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene -glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between –248 to –108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C₂H₄ and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C₂H₄ and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C₂H₄ in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C₂H₄ was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C₂H₄. However, significant C₂H₄-induced activity was obtained with a promoter fragment containing the AT and PR elements together.     

Selection of penstemide and serrulatoloside producing clones in penstemon serrulatus by small-aggregate cloning

A plating method is described for cultured cells of Penstemon serrulatus Menz. which allows effective cloning of aggregates consisting of less than 10 cells, on a simple synthetic medium. The obtained clones snowed high diversity with respect to their ability to produce penstemide, an ester iridoid of the Valeriana-type. Some of the clones produced higher quantities of this compound than the original cell line. The content of another iridoid glucoside of the same type, serrulatoloside, was also determined.Abbreviations SH Schenk and Hildebrandt (1972) - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - PE plating efficiency - d.w. dry weight - sd standard deviation     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

Summary A soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with -d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.     

Factors Affecting the Resistance of Lactobacillus fermenti to Lysozyme

The sensitivity of Lactobacillus fermenti ATCC 9338 to lysozyme has its peak during the exponential phase of growth, after the autolytic activity of the organism has begun to decline. Cells from the stationary growth phase are resistant to lysozyme. The two lytic activities require different ionic conditions for their functioning; they appear mutually exclusive. Incubation with trypsin renders cells from all growth phases sensitive to lysozyme. The effect of trypsin is independent of the presence of lysozyme and vice versa, as demonstrated by use of trypsin inhibitors. Cells from early stationary phase of growth require higher temperature for optimum lysozyme action than do those from the exponential growth phase.     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

Cytological study of inhibitory effects of IAA on the root growth in the Pinus silvestris

It has been found that IAA at the concentrations 0.1--10.0 ppm retards the pine root growth and decreases mitotic activity. All the applied concentrations of this hormone cause a decrease of the 3H-thymidine incorporation index and inhibit endomitotic polyploidization in suprameristematic segments. The mean time of the cell cycle prolongs and the heterogeneity of cellular populations increases in parallel with the increase of IAA concentration. The template activity of DNA decreases under the influence of the applied concentrations of IAA; this effect is being particularly strong in the meristematic root segment. IAA exerts also an inhibitory influence on protein synthesis, especially reducing the synthesis of histones.     

Biotransformation of hydroquinone to arbutin in plant <Emphasis Type="Italic">in vitro</Emphasis> cultures — preliminary results

Cells from Echinacea purpurea (L.) Moench. (Asteraceae), Exacum affine Balf. f. (Gentianaceae), Melittis melissophyllum L. (Lamiaceae), Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. (Rutaceae) agitating cultures perform a biotransformation reaction on exogenously supplied hydroquinone into its β-D-glucoside — arbutin, product with valuable medicinal and cosmetic properties. The maximum content of arbutin (determined by HPLC) in the biomass from investigated cultures is 4.01; 3.44; 1.79; 2.48 and 5.07 g/100 g d.w., respectively. Nothing but Ammi majus L. (Apiaceae) cultures contain trace amounts of the product. Arbutin is accumulated in cells; it is occasionally found in media only in vestigial amounts. In most of the investigated cultures the efficiency of the biotransformation process is about 60 %.