Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

Amylolytic Activity in Taproots of Diploid and Tetraploid Medicago sativa L.

Our objective was to determine whether starch degradation intaproots of alfalfa (Medicago sativa L.) after defoliation wasassociated with activity and isoform complement of endo- andexo-amylases. Taproots of genetically comparable diploid (2x)and tetraploid (4x) populations and the tetraploid cultivarHi-Phy were recovered immediately after defoliation and at approx.4-d intervals thereafter. Taproot tissues were analysed forstarch concentrations and activities of endo- and exo-amylases.An electrophoretic blotting technique was used to examine amylaseisoforms. Starch degradation was most rapid in taproots of Hi-Phy,slowest in taproots of the 2x population, with the 4x populationbeing intermediate. The 4x population had a greater initialincrease in endo-amylase activity compared to the 2x population;however, Hi-Phy averaged eightfold greater endo-amylase activitythan either 2x or 4x populations. Although exo-amylase specificactivity was at least 500-fold greater than endo-amylase specificactivity in all populations, changes in endo-amylase activitywere more closely associated with trends in starch degradation.Multiple isoforms of endo- and exo-amylase were observed intaproots of all populations. Taproots of Hi-Phy contained anendo-amylase isoform that was not apparent in the 2x or 4x populationsthat may contribute to the high activity of this amylase intaproots of this cultivar. These results, although correlative,suggest an important role for endo-amylase in taproot starchhydrolysis after defoliation. Medicago sativa (L.), alfalfa, taproots, herbage, diploid, tetraploid, starch, endo-amylase, exo-amylase, isoforms, electrophoresis     

Radiosensitivity of Mammalian Cells: I. Timing and Dose-Dependence of Radiation-Induced Division Delay

The time of onset and duration of division delay induced by exposure to 250-kvp x-irradiation have been measured in several mammalian cell lines grown in suspension culture. Unique times of action (i.e. interval from irradiation to cessation of division) late in G₂ are characteristic for HeLa, L-5178Y, and Chinese hamster cells, and the time of action is independent of dose over the range 25-800 rads. The duration of delay was directly proportional to dose; all irradiated cells divided at least once and maintained their relative positions in the life cycle for periods exceeding one generation time. Neither random nor synchronous cultures exposed at varying times in the life cycle exhibited differences in radiation sensitivity measured either by onset or duration of the delay period. The time of action was experimentally indistinguishable from the point marking completion of protein synthesis essential for division, leading to speculation that division delay involves a translation defect.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Multiplicity of Steady States in Glycolysis and Shift of Metabolic State in Cultured Mammalian Cells

Cultured mammalian cells exhibit elevated glycolysis flux and high lactate production. In the industrial bioprocesses for biotherapeutic protein production, glucose is supplemented to the culture medium to sustain continued cell growth resulting in the accumulation of lactate to high levels. In such fed-batch cultures, sometimes a metabolic shift from a state of high glycolysis flux and high lactate production to a state of low glycolysis flux and low lactate production or even lactate consumption is observed. While in other cases with very similar culture conditions, the same cell line and medium, cells continue to produce lactate. A metabolic shift to lactate consumption has been correlated to the productivity of the process. Cultures that exhibited the metabolic shift to lactate consumption had higher titers than those which didn’t. However, the cues that trigger the metabolic shift to lactate consumption state (or low lactate production state) are yet to be identified. Metabolic control of cells is tightly linked to growth control through signaling pathways such as the AKT pathway. We have previously shown that the glycolysis of proliferating cells can exhibit bistability with well-segregated high flux and low flux states. Low lactate production (or lactate consumption) is possible only at a low glycolysis flux state. In this study, we use mathematical modeling to demonstrate that lactate inhibition together with AKT regulation on glycolysis enzymes can profoundly influence the bistable behavior, resulting in a complex steady-state topology. The transition from the high flux state to the low flux state can only occur in certain regions of the steady state topology, and therefore the metabolic fate of the cells depends on their metabolic trajectory encountering the region that allows such a metabolic state switch. Insights from such switch behavior present us with new means to control the metabolism of mammalian cells in fed-batch cultures.     

Translocation in Sugar-beet: I. ASSIMILATION OF 14CO2 AND DISTRIBUTION OF MATERIALS FROM LEAVES

¹⁴CO₂ was supplied to leaves, and movement of labelled carbonto other parts of the plant was assessed. Young growing leavesutilized assimilated carbon for their own growth and did notexport carbon to the rest of the plant, while fully expandedleaves exported much of their photosynthate, both to root andto young leaves. Translocation from a particular leaf was tothe two or three younger leaves on the same side of the plant,and to a sector of root below the source leaf. Specific distributionto growing leaves could be modified by partial defoliation.There was no movement of material to leaves which had emergedbefore the source leaf. Part of the carbon entering a leaf by assimilation (and, foryoung leaves, by translocation) was incorporated into insolublematerial, especially in young leaves. Some of the carbon enteringa developing root was permanently stored as sucrose, althoughmuch also entered insoluble material. Loss from the leaf ofcarbon fixed during a short period of photosynthesis was rapidat first but continued at a decreasing rate for several days.Some carbon fixed into the insoluble fraction was translocatedfrom the leaf later, during senescence. Sucrose was the mainmaterial translocated immediately after photosynthesis.     

Amino Acid Transport into Cultured Tobacco Cells: II. EFFECT OF CALCIUM

The effects of calcium ions on lysine transport into cultured Wisconsin-38 tobacco cells were examined. In the presence of calcium, lysine was transported at a relatively low rate for 30 to 40 minutes followed by a period of increasing rates and subsequent stabilization at a higher rate after 2 to 3 hours. In the absence of calcium, transport was uniformly low.     

A Comparison of Survival and Repair of Uv-Induced DNA Damage in Cultured Insect VERSUS Mammalian Cells

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10–50 J/m² UV irradiations.     

Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and β-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.     

Hybridization of Senecio squalidus and S. viscosus and Introgression of Genes from Diploid into Tetraploid Senecio Species

Natural triploid hybrids (Senecio x londinensis Lousley) betweenS. squalidus L. (2n = 20) and 5. viscosus L. (2n = 40) are fairlyfrequently found in Britain. Under glasshouse conditions bothnatural and artificial hybrids displayed very low levels ofseed fertility and gave rise to morphologically diverse F₂ plantsat about the triploid or pentaploid chromosome levels. By theF₄ generation, progeny of a F₂ pentaploid plant had somaticchromosome numbers near to the tetraploid level and considerablyincreased pollen and seed fertilities. Such fertile tetraploidsegregants of S. x londinensis permit the introgression of S.squalidus genes into S. viscosus, and may indicate the courseof introgression into other tetraploid species of Senecio. Senecio, hybridization, introgression     

Boron Nutrition of Cultured Tobacco BY-2 Cells: I. Requirement for and Intracellular Localization of Boron and Selection of Cells that Tolerate Low Levels of Boron

Cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow2) grown under the standard culture conditions (1 mg boron liter^–1medium as boric acid) contained boron at a concentration of2.26 mg boron kg^–1 oven-dried cells and the protoplastcontained 1.26% of the boron in the cells. The cells requiredboron for growth and the half-maximum growth rate was obtainedwith 0.056 mg of boron liter^–1 medium. Subculturing thecells in media with lower concentrations of boron allowed selectionof cells that can grow even in the presence of 1 µg boronliter^–1 medium. Cell walls of the selected cells seemedto be thicker than those of the control cells and Golgi bodieswere accompanied by more secretory vesicles than those in thecontrol cells. (Received May 25, 1992; Accepted September 10, 1992)     

Dormancy of Rice Seed: I. THE DISTRIBUTION OF DORMANCY PERIODS

The mean dormancy period is defined as the mean time taken fromharvest for the individual seeds of a population to attain theability to germinate under normal conditions. Evidence is presentedwhich shows that the distribution of dormancy periods amongthe individual seeds of a pure line is normal. Since germinationusually reaches 100 per cent., and because a normal distributionis symmetrical, a good estimate of the mean dormancy periodcan be obtained by observing the time taken from harvest tothe point when 50 per cent. of the population is capable ofgermination as shown by a series of germination tests. The mainerror involved is due to the fact that there is no obvious wayof deciding, to within a few days, when grain is ripe for harvest.Harvesting the seed prematurely tends to speed up the processesleading to loss of dormancy whereas late harvesting has littleinfluence on the date on which the mean dormancy period is achieved;it follows that when estimating the mean dormancy period theerror is much smaller if seed is harvested too early ratherthan too late. Contrary to some previous reports, no correlation has been detectedbetween ‘duration’ (period from sowing to modalflowering plus 30 days) and mean dormancy period among differentvarieties.     

Response of Cultured Mammalian Cells to Diphtheria Toxin III. Inhibition of Protein Synthesis Studied at the Subcellular Level

Diphtheria toxin inhibited protein synthesis in intact KB cells. The action of the toxin upon the cell did not result in disaggregation of polyribosomes, or in impairment of their ability to function in protein synthesis. A reduction in single ribosomes and a concomitant increase in polyribosomes did result from the action of toxin. Nascent peptides were not cleaved from polyribosomes by the action of toxin, but treatment of fully intoxicated cells with puromycin resulted in cleavage of these peptides, and caused accelerated polyribosome breakdown. Our data indicated that the toxin must enter the cell to exert its effect. The component or components sensitive to toxin were localized in the 100,000 x g supernatant fraction of cytoplasmic extracts. When extracts from intoxicated cells were treated with nicotinamide, a significant proportion of their capacity to synthesize protein was restored. The specificity of this reaction suggested that nicotinamide adenine dinucleotide is involved in the action of toxin in the intact cell, and that one component inactivated by toxin is soluble transferase II.     

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.     

Adhesion and Interiorization of Trypanosoma cruzi in Mammalian Cells1

A quantitative method for experimentally separating the adhesion and interiorization phases of the interaction of Trypanosoma cruzi with mammalian cells was developed. Incubation of confluent monolayers of mammalian cells with epimastigotes or trypomastigotes at 4°C allowed the evaluation of the number of adhered parasites that do not become interiorized at this temperature. Quantification of interiorized parasites at 34°C was achieved by employing hypotonic lysis to eliminate the extracellularly adhered trypomastigotes. Both adhesion and interiorization were found to be proportional to the time of exposure of cells to parasites and to the multiplicity of infection. These phenomena occur normally for trypomastigotes in the absence of serum with LLC-MK₂ cells, HeLa cells, and 3T3 fibroblasts. Moreover, it was possible to obtain trypomastigotes that presented the same infectivity to LLC-MK₂ cells as did parasites obtained in the presence of 2% fetal calf serum after 10 serial passages in a medium devoid of serum. Inhibition of adhesion (of epimastigotes and trypomastigotes) and of interiorization (of trypomastigotes) was obtained with inactivated normal serum from several sources, a saturation effect being observed at a final concentration of 20%. Bovine serum albumin, at the concentrations present in the sera, had no inhibitory effect. Trypomastigotes that have been pre-incubated with 40% FCS (45 min at 4°C) showed decreased adhesion and interiorization indices, effects that can be reversed by trypsinization of the parasites prior to exposure of the cells. A progressive internalization of previously attached trypomastigotes was observed on raising the temperature from 4°C to 34°C; no spontaneous detachment of parasites was detected up to 120 min. Approximately 75% of the adhered parasites were found inside the cells after 45 min at 34°C. The presence of normal inactivated calf serum during incubation at 34°C resulted in a certain degree of detachment and in a lower interiorization index.     

Fertility in Crosses Involving Diploid and Autotetraploid Raspberries: I. The Embryo

Crosses between diploid raspberry cultivars (Rubus idaeus L.)and their autotetraploid forms have shown that embryo shapedepends on three main factors—the variety, the stage ofdevelopment reached before growth ceases, and the ploidy ofthe embryo itself. Embryo growth, however, depends on the abilityof the endosperm to nourish the embryo and so on the harmoniousco-existence of derivatives of the gametes which formed theendosperm. Crosses between plants of unequal ploidy produceendosperms which are unbalanced. In general, the order of fertilityof the crosses is 2n selfed, 4n selfed, 4n X 2n, 2n X 4n, andthis can be partly explained if the effect of chromosome doublingis to increase the genetic strength of the male gametes morethan that of the female. Variations in the embryo growth inthe 2n X 4n and 4n x 2n crosses are ascribed to smaller differencesin genetic strength between varieties and between the male andfemale gametes within a variety. Embryos derived from crosseswithin a single variety grew less well than those derived fromcrosses between varieties, other factors being equal, and thisis considered to be an early expression of inbreeding depressionin the embryo.     

Organization and Evolution of the Mammalian Genome: I. Polymorphism of H-2 Linked Loci

To test the hypothesis that the H-2 polymorphism is adaptive, the degree of polymorphism of loci linked to the H-2 complex on chromosome 17 of the house mouse was compared to the degree of polymorphism of loci located on other chromosomes. Published theoretical analyses show that polymorphisms subject to natural selection usually reduce the polymorphism of linked neutral loci. The first test of the hypothesis was based on data obtained from a survey of the polymorphism of 12 isozyme-encoding loci in wild house mice from Europe, North Africa and South America. Results of this test showed that, on the average, H-2-linked loci were as polymorphic as loci located on other chromosomes. In fact, the data suggested that H-2 linked loci might be more polymorphic than other loci. To test this hypothesis more rigorously, data for the 12 isozyme-encoding loci were augmented with data from published surveys of the polymorphisms of 59 loci in house mice from Europe and North America. Results of these tests showed that polymorphic loci linked to the H-2 complex tended to be more, rather than less, polymorphic than loci located on other chromosomes. The cluster of highly polymorphic loci seems to be related to linkage of these loci to the highly polymorphic H-2 complex, but the way in which the influence is exerted could not be readily explained.