Organic Nutrients in the Media for Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on media containing different organic nutrients. Of the sugars tested sucrose was better than maltose, glucose and fructose, and sucrose had an optimum concentration of 3 to 4 %. D-Mannose was significantly less effective than the other sugars. The amino acid mixtures casamino acids (casein hydrolysate) and tryptone increased growth while yeast extract was inhibitory and malt extract without effect. Optimal concentrations were 2 to 3 g · l^-1 casamino acids and 3 to 4 g · l^-1 tryptone. It was to some extent possible to substitute the amino acid mixtures with a single amino acid (glutamine at 300 mg · l^-1). Arginine was inhibitory and asparagine was without any effect. Vitamins proved to be unnecessary although there was a tendency towards increased growth with nicotinic acid and meso-inositol. Purines and pyrimidines were added to the medium but with no effect. Liquid endosperm from coconuts (10 to 15%) increased growth while the liquid endosperm from Aesculus hippocastanum was inhibitory. On the basis of these results a revised medium is proposed for the in vitro propagation of Cymbidium.     

Screening <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (Spirulina) strains for heterotrophy

Thirty-five clonal, axenic Arthrospira strains were screened for their ability to grow heterotrophically on six carbon sources (20 mM). Glucose (34 strains) and fructose (24 strains) were the only substrates permitting growth in the dark. In some assays, however, not every replicate grew and, in at least one strain (D867), repeat assays over 2 years on material maintained in the light indicated that the ability to use fructose in the dark had become lost. Four further strains from other culture collections were compared, because they are presumed duplicates of three of the main set of strains; in at least three cases the duplicates of a pair differed in their ability to use fructose in the dark. In another comparison, where straight and helical morphotypes of the same strain could be compared, two of the four straight morphotypes (including one duplicate strain) grew with glucose in the dark, whereas none of the helical morphotypes did so. It is suggested that genetic drift has led to losses in the ability to grow heterotrophically in some strains.Ten of the main set of strains were tested for their ability to grow photoheterotrophically with four of the carbon sources (glucose, maltose, fructose, sucrose). All grew with glucose and maltose, but none with fructose or sucrose, in spite of the fact that eight (of this subset) grew with fructose in the dark. Sucrose led to most of a culture lysing, but often with short fragments of trichome surviving and subsequently giving rise to a normal culture. All the surviving cells in the transitory stage showed the presence of large intrathylakoidal granules, which had disappeared by the time that the culture had returned to a healthy state. Bleaching and recovery were more rapid at 70 than 10 μmol photon m⁻² s⁻¹. The presence of DCMU prevented this recovery. There was no bleaching when an inoculum of the recovered material was subcultured to medium with sucrose.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

Regulation of biosynthesis of bacilysin byBacillus subtilis

Summary Production of the dipeptide antibiotic bacilysin byBacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6×10^–4M.     

Sugar uptake by maize endosperm suspension cultures

Maize (Zea mays L.) endosperm suspension cultures are a useful model system for studying biochemical and physiological events in developing maize endosperm. In this report, sugar uptake by the cultures is characterized. Uptake of ¹⁴C-labeled fructose and l-glucose was linear with time, while the rate of uptake of radioactivity from sucrose increased over a 120 min period. Both saturable and linear components of uptake were observed for fructose, glucose, sucrose, 1′-deoxy-1′-fluorosucrose, and maltose. Uptake of mannitol, sorbitol, and l-glucose took place at lower rates and was linear with concentration. Rates of incorporation of radioactivity from fructose and glucose exceeded that of sucrose at all concentrations tested. Kinetics of 1′-deoxy-1′-fluorosucrose uptake indicated that ¹⁴C from sucrose can be taken up by a saturable carrier of intact sucrose as well as by invertase hydrolysis and subsequent uptake of hexoses. Cell wall invertase was demonstrated histochemically. Further study of fructose uptake at a concentration at which the saturable component predominated revealed sensitivity to metabolic inhibitors, respiratory uncouplers, the nonpermeant sulfhydryl reagent p-chloromercuribenzenesulfonic acid, and nigericin. Uptake was not affected by valinomycin plus K⁺ and was stimulated by fusicoccin. Fructose and glucose uptake was not pH-sensitive below pH 7.0, whereas uptake of radioactivity from sucrose and 1′-deoxy-1′-fluorosucrose declined as the pH was increased above 5.0. Fructose uptake was not completely inhibited by glucose and vice versa, suggesting the presence of specific carriers. These results indicate that maize endosperm suspension cultures (a) absorb fructose via a typical, energy-requiring, carrier-mediated proton cotransport system; (b) possess saturable carriers for glucose and sucrose; and (c) also absorb sucrose via hexose uptake after sucrose hydrolysis by extracellular invertase.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Exchange of Metabolites in Cyanophora paradoxa and its Cyanelles*

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic CO₂-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert ¹⁴C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for ¹⁴C-labelled sorbitol and ³H₂O were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements of the solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvay's salts at half-strength containing 1 g l^-1 glutamine, 1 g l^-1 casein hydrolysate, 7.5 M abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.     

Rubratoxin production by penicillium rubrum when grown in a synthetic medium containing different sources of carbon and nitrogen

A sterile mineral salts broth was fortified with different additives, inoculated with conidia ofPenicillium rubrum P-13, and incubated quiescently for 14 days or with shaking for 3 to 5 days. Maximal fungal growth and rubratoxin production occurred when the broth contained 20% sucrose. Broth with 10% glucose, 10% fructose, 5% maltose, or 1% asparagine supported formation of substantial amounts of rubratoxin (52.9–78.5 mg/100 ml). When the broth was fortified with glucose plus lysine, arginine aspartic acid, cystine, ammonium citrate, or ammonium phosphate, moderate amounts (27.5–39.5 mg/100 ml) of rubratoxin and mycelium (0.1–1.5 g/100 ml) were produced. Presence in the broth of 5% galactose or starch resulted in accumulation of small amounts (22.2 and 24.6 mg/100 ml, respectively) of rubratoxin and mold tissue (0.70 and 0.5 g/ 100 ml, respectively). Whereas some toxin was recovered from mineral salts broth fortified with lactose or ribose, toxin was not recovered when the mold grew in broth containing mannitol or fumarate. With the exception of gluconate which supported some growth and toxin formation and ethanol which permitted formation of small amounts of toxin, other carbon sources resulted in little or no fungal growth and no toxin formation. Yields of rubratoxin decreased with an increase in amount of agitation or length of incubation ofP. rubrum cultures. Mold growth increased and toxin formation decreased with an increase in volume of culture.     

Factors affecting lipase production in Syncephalastrum racemosum

Syncephalastrum racemosum grown as a static culture showed maximum lipase production at 30°C in 2d at pH 8.0. When the medium was supplemented with fructose, maximum production of lipase per unit of growth was achieved, followed by raffinose, sucrose, ribose, galactose, maltose, lactose, mannitol and glucose. Amongst the nitrogen sources tested, corn steep liquor at 8% (v/v) produced maximum enzyme; there was evidence of catabolite repression by glucose when groundnut protein, soybean meal, milk casein or wheat bran were the sources of nitrogen. Calcium, potassium and sodium citrates, each at 0.1% (w/v), increased the yield of lipase.     

Effect of Low Levels of Calcium on Exudation of Sugars and Sugar Derivatives from Intact Peanut Roots under Axenic Conditions

The effects of 10, 20, 35 and 50 mg of Ca²⁺ per liter on the qualitative and quantitative exudation of sugars from roots of 5-week-old peanut plants, Arachis hypogaea L., grown axenically in nutrient solutions, were measured. Nutrient solutions in which plants had been growing were collected at weekly intervals for 4 weeks, sugars in them were measured by gasliquid chromatography of the trimethylsilyl derivatives. Arabinose, ribose, xylose, fructose, mannose, glucose, galactose, mannitol, galacturonic acid, inositol, sucrose, and five unknowns were found. Qualitative and quantitative differences in exudates were correlated with age of the plants and calcium level. Four times more sugar was exuded at 10 mg than at 50 mg of Ca²⁺ per liter but no significant differences in growth were observed. Ion efflux measurements suggested that low levels of Ca²⁺ increased root cell membrane permeability.     

Inhibitory Effect of Carbohydrate on Flowering in Lemna perpusilla. I. Interaction of Sucrose With Calcium and Phosphate Ions

Flowering in Lemna perpusilla 6746 grown on tenth-strength Hutner's medium under short days was inhibited by 30 mM sucrose, glucose or fructose, but not by mannitol. The inhibition by sucrose does not appear to be due to sucrose-induced acidification of the medium during growth, or to trace metal contaminants of the sugar. Inhibition was partially prevented by raising either Ca²⁺ or phosphate to levels used in half-strength medium. Possible mechanisms for these effects are discussed.     

Trophic dependencies of some larval chironomidae (Diptera) and fish species in the River Thames

Experiments are described to characterise the heterotrophic potential of Westiellopsis prolifica Janet, which fixes nitrogen under light and dark conditions. The growth of the organism in terms of dry weight increase, was more in fructose, lactose, sucrose, sorbose, galactose, glucose, sodium acetate, mannitol, sorbitol, glycerol, ethyl alcohol and butyl alcohol, when the alga was pretreated with light and subsequently incubated with the substrates in light. Mannose, xylose, acetic acid, propionic acid, fructose 1,6 di Po₄, pyruvic acid, dihydroxyacetone and succinic acid decreased the growth of the organism in the same condition. In dark incubation after pretreatment with light, as well as in the dark, Westiellopsis showed a better growth response to almost all the exogenous substrates. However, after pretreatment either with light or dark, the test organism utilised exogenous substrates quicker in light than in dark incubations. These experiments would suggest that the substrate specificity and efficiency of substrate utilisation by the alga during its heterotrophic growth are governed by the growth conditions.     

Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnifolium Cav.

The effect of inoculum size, carbon sources (fructose, glucose, maltose, sucrose), nitrate and ammonia on solasodine production by Solanum eleagnifolium Cav. was studied. The specific growth rate was estimated to be 0.15–0.20 d^-1 with all sugars tested at a concentration of 90 mM. Sucrose (180 mM) produced the highest biomass value (about 2.8 mg DW ml^-1) while the lowest one was produced by maltose. Although solasodine productivity values after 11 days of culture were similar for all sugars tested, the maximum values of productivity (0.9 mg g^-1 d^-1) were achieved after 6 days of culture with sucrose (180 mM). Solasodine productivity of cultures conducted with a large inoculum (20% w/v fresh material) was double that with a small inoculum (10% w/v fresh material).     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

The effect of different carbohydrate sources upon the initiation of embryogenesis from barley microspores

Isolated microspores of Hordeum vulgare L. cv. Igri were incubated in the presence of different sugars. In the presence of maltose, the optimum concentration for the development of embryoids or calluses from the microspores was 175 mM. At this concentration 0.2% of the cells developed into embryoids or calluses. Microspores cultured without a carbohydrate source died after three days' incubation. In contrast microspores incubated in the presence of sucrose, glucose or fructose died within three days. Moreover, microspores also died when incubated in the presence of a combination of 175 mM maltose with varying concentrations of either sucrose, glucose or fructose. It is concluded that incubation of microspores in the presence of sucrose, glucose or fructose results in the death of the cells via some unknown mechanism. In contrast to this, maltose can sustain development of embryoids and calluses from cultured microspores.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

A method for the isolation of Chinese hamster cell variants with an altered ability to utilise carbohydrates

Chinese hamster ovary cells (CHO-K1) are able to utilise only a few carbohydrates for growth such as glucose, mannose, fructose and galactose. They do not grow on ribose, lactose, sucrose, glycerol, lactate, pyruvate, citrate, succinate, fumarate or malate nor on glycogenic or ketogenic amino acids. After mutagenesis and selection in glucose free medium supplemented with various, individual, growth substrates, we have isolated single-cell derived clones which are now able to grow on one of the following energy source: ribose, lactose, sucrose or lactate.