REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

MATURATION OF ALGAL ZYGOTES: ALTERNATIVE EXPERIMENTAL APPROACHES FOR CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Young zygotes from crosses of Chlamydomonas reinhardtii Dang. mutant and wild-type strains were incubated, in the presence or absence of light and/or nitrogen to determine whether continuation of conditions inducing gamete formation permits zygospore formation without loss of viability. Different culture media, continuous illumination vs. dark incubation and various durations of the maturation period were tested, for effect on zygospore germination efficiency, zygospore “burst size” and zoospore viability. Following either the routine maturation procedure of dark incubation on standard minimal medium, or following a new procedure of incubation under continuous illumination on N-free medium, zygospore formation can be ensured and high germination efficiencies obtained within 3 days after mating. Tetrad analysis indicates meiosis occurs normally whether zygotes have been matured in the presence or absence of light or nitrogen. Preliminary data suggest an effect of increased maturation time on the transmission of cytoplasmic genes, if a N-free continuous illumination maturation protocol is followed. Two experimental approaches for the maturation of C. reinhardtii zygotes are suggested and advantages of each are discussed.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL^?1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell^?1) over a 5-fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient-replete medium, even with a significant reduction in growth rate due to non-optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]     

The Uptake of Phosphate by Plants from Flowing Nutrient Solution: IV. EFFECT OF PHOSPHATE CONCENTRATION ON THE GROWTH OF TRIFOLIUM REPENS L. SUPPLIED WITH NITRATE, OR DEPENDENT UPON SYMBIOTICALLY FIXED NITROGEN

Breeze, V. G. and Hopper, M. J. 1987. The uptake of phosphateby plants from flowing nutrient solution. IV. Effect of phosphateconcentration on the growth of Trifolium repens L. suppliedwith nitrate, or dependent upon symbiotically fixed nitrogen.—J.exp. Bot. 38: 618–630. Nodulated white clover plants were subjected to a range of phosphateconcentrations in flowing solution culture (0.32 to 8.0 mmolm^–3 P) at 41 d from sowing, either supplied with nitrateor dependent on symbiotically-fixed nitrogen. No effect of phosphateconcentration in solution on dry matter production, relativegrowth rate, root/shoot ratio, or water soluble carbohydrateconcentration of the plant tissue was observed after 24 d fromthe start of the experiment, although the plants supplied withnitrate yielded more than the others. Phosphate uptake throughoutthe experimental period was related to the solution concentration,but the source of nitrogen did not affect the phosphorus concentrationsof the shoots. However, the roots of the plants dependent onsymbiotically-fixed nitrogen had higher concentrations of phosphorusthan those supplied with nitrate, but this did not appear tobe due to an increased phosphorus requirement for nitrogen fixation,because the amount fixed was unaffected by the phosphate concentrationin solution. The cation-anion balance showed that plants dependenton nitrogen fixation had no larger requirement for calcium thanplants supplied with nitrate, but a requirement for hydroxylions equivalent to over 130 kg lime per tonne of dry shoot.It is suggested that the enhanced phosphate uptake by plantsdependent on nitrogen fixation is due to this need for a cation-chargebalancing anion. Key words: Phosphate uptake, nitrogen fixation, Trifolium repens L., repens L., cation-anion balance, flowing solution culture     

Effects of Nutritional and Hormonal Factors on the Formation of Anti-plant-viral Protein by Mirabilis jalapa L. Cells in Suspension Culture

To obtain basic information of the production of anti-plant-viral protein by Mirabilis jalapa cells in suspension culture, the cultural conditions, especially the nutritional ones, for MAP formation and cell growth were investigated. It was found MAP formation was greatly repressed by a high concentration of 2,4-D and the addition of cytokinin. The concentration of nitrogen sources, phosphate, and CaCl₂ had marked effect. Revised medium was prepared due to the corresponding optimum concentration obtained from our investigations. MAP productivity in the revised medium became twice that in the basal medium. Changes in MAP content were also examined during successive subculturing. It was recognized that MAP content increased in the early generations, then decreased slowly during subculturings.     

Influence of Nitrogen and Sucrose in the Medium and of Irradiance of the Stock Plants on Root Formation in Pelargonium Petioles Grown in vitro

In vitro root formation was studied in petiole explants from Pelargonium×hortorom Bailey cv. Radio exposed to different irradiance (2.5. 11.6 or 23.0 W/m²). Optimal root formation was found when stock plants were subjected to 2.5 W/m² and explants were grown on a medium with 20 g/I of sucrose and 5.0 mM of nitrogen. The number of roots per explant decreased with increasing nitrogen or sucrose concentrations in the growth medium and with increasing irradiance applied to the stock plants. Variation in temperature (17, 21 or 25°C) did not affect root formation. The number of roots per explant was higher in explants from young petioles than in explants from older petioles. The initial nitrogen content of the explants was highest at the lowest irradiance. The content of reducing sugars and sucrose increased with increasing irradiance. The endogenous levels of lysine, arginine and ornithine were highest at 2.5 W/m² and the levels of proline. alanine, γ-aminobutyric acid, glutamic acid, glutaminc, aspartic acid and asparagine were highest at 23.0 W/m². With rising nitrogen concentration in the medium, the final endogenous content of nitrogen in the explants increased, whereas the content of reducing sugars decreased. When the sucrose concentration in the medium increased, the endogenous content of reducing sugars rose, and the nitrogen level was lowered. Variations in the nitrogen concentration in the growth medium influenced the final level of free amino acids in the explants.     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Influence of carbon and nitrogen sources on Flavobacteriumgrowth and zeaxanthin biosynthesis

Growth and production of zeaxanthin by Flavobacterium sp were studied using different carbon and nitrogen sources in a chemically defined medium. The best growth was supported by sucrose, but glucose yielded similar carotenoid concentrations. Both asparagine and glutamine stimulated growth and pigment formation. Carotenoid production and glucose consumption increased as a function of asparagine concentration. In the presence of asparagine, high glucose concentrations decreased pigment production without affecting biomass formation. In the absence of glucose, asparagine could not support growth and zeaxanthin production. When compared to the effect of 55 mM glucose, 10 mM oxaloacetate increased growth and carotenoid production. Pyruvate and other intermediates of the citric acid cycle showed a similar stimulatory effect. The intermediates of glycolysis: glucose 6-phosphate and fructose 1,6-diphosphate did not support growth. These results suggest that Flavobacterium sp utilizes asparagine primarily as a nitrogen source for growth and production of zeaxanthin. Received 29 September 1998/ Accepted in revised form 23 April 1999     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.     

Growth responses and root porosity of rice in relation to temperature,light intensity and aeration

Summary Experiments were conducted under which rice plants were grown under three energy input levels (temperature and light) and two solution-oxygen concentrations. The rice variety was Colusa. Increasing the energy input increased total top growth. There was no significant effect of solution-oxygen concentration on top growth. The highest number of tillers developed under medium energy input followed by high energy input and the least under low energy input. There was a trend towards more tiller production under low oxygen conditions as compared to high oxygen. Decreasing the oxygen supply increased the dry weight production of roots under the two higher energy input levels, but had no significant effect under the low energy input. The highest amount of root growth occurred under high and medium energy input and least under lower energy input. Increasing the energy input increased the percent root porosity, but the oxygen level had no significant effect on root porosity. None of the treatments had a significant effect on nitrogen concentration in the tops. The amount of water necessary per unit dry matter production had a trend towards increase with increased energy input level and also significantly higher under low oxygen as compared to high oxygen conditions. Contribution of the Department of Soils and Plant Nutrition, University of California, Riverside. Supported in part by NSF Grant No. GB-5733X1.     

Effects of chemical components and nitrogen sources on zygospore development inPhycomyces blakesleeanus

We examined the effects of chemical components and nitrogen sources on zygospore development, using 62 different ingredients based on Sutter's synthetic medium Sl, which has been widely used for studies of sexual physiology inPhycomyces. An increase of inorganic microelements such as ZnSO₄, NaMoO₄ and CaCl₂ promoted an increase in the number of zygospores per unit area. Glutamate (Glu) contained in Sl as the sole nitrogen source was indispensable for sexual development, and replacement of Glu with NH₄ ⁺ (Am) strongly inhibited it, mainly because of growth inhibition. However, zygospore production was enhanced 1.8-fold by equivalent amounts of both Glu and Am as compared with Glu alone. A newly developed medium, mSl+Am, enriched with Am and the above-mentioned effective microelements doubled the number of zygospores formed per unit area (density), compared with Sutter's original Sl, and increased both the density and the weight (volume) of zygospores, 1.6- and 2-fold, respectively, compared with potato-dextrose-agar medium enriched with yeast extract and casitone (PDAYC). Sexual stimulation by mSl+Am was also observed in the mating of a pair of β-carotene-deficient mutants. Methionine sulfoxime, an inhibitor of glutamine synthetase, strongly inhibited the progress of mating without significant growth inhibition. The first and the second authors contributed equally to this work.     

Production of Proteases from Brevibacterium Linens

Protease formation in submerged cultivations of Brevibacterium linens was studied. The effect of several proteinaceous materials on the production of proteolytic enzymes was investigated in mineral media containing 0.2% malt extract for bacterial growth. The addition (0.5%) of yeast extract or enzymatically hydrolyzed casein considerably increased the amount of protease formed, whereas ammonium salts supplied additionally in most cases had a repressive effect on enzyme formation. Furthermore, the kinetic of protease formation was determined in a highly instrumented fermenter system. Respiration activity indicated several phases of bacterial growth. Most of the proteolytic activity was synthesized during active growth; there was only a small increase in the stationary phase. A total proteolytic activity of 36 U/ml was formed in 24 hr. Concentration of α-amino nitrogen decreased steadily and ammonium ions accumulated during bacterial growth. Electrophoretic analysis revealed the occurrence of one leucine aminopeptidase (26 kDa monomer) and several proteases. There is a broad spectrum of proteolytic active proteins in the range of 11-66 kD which may be caused by some auto-degrading effects.     

Biosynthesis of extracellular low-molecular-mass papain and trypsin inhibitors by Streptomyces sp. 22: effect of cultural conditions

The production of extracellular inhibitors of papain and trypsin by Streptomyces sp. 22 was studied under different cultural conditions including complex and defined media, temperatures ranging from 18 °C to 37 °C and a variety of sole carbon and nitrogen sources. In complex nutritionally rich medium, maximal specific growth rates were obtained at 37 °C, whereas the highest specific production rates for both papain and trypsin inhibitors were registered at 18 °C. Studies on the effect of different carbon and nitrogen sources in defined media underline the importance of the nitrogen source as a strong regulator of the biosynthesis of both inhibitors. Enhanced formation of the inhibitory compounds occurred in the presence of casein. The dynamics of the formation of both inhibitors in defined media showed close association with growth. However, a partial separation of production phases for papain and trypsin inhibitors was observed in complex medium. The results imply differences in the regulation of biosynthesis of the two inhibitors.     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.     

Effect of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates of Phoma bakeriana

The effects of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates (PB1, PB2, PB3) of Phoma bakeriana were investigated in vitro. There was no isolate effect on growth and pycnidium production but some evidence of a medium effect. Exceptionally high growth rate on two media corresponded with high pycnidium production; while low growth rates on other media were generally associated with high sporulation. There was a large temperature effect and an isolate effect on growth with a significant temperature by isolate interaction. Temperatures of 5°C, 10°C and 35°C inhibited pycnidium formation completely while the range 15–30°C had no effect on sporulation. Maximum growth and pycnidium production occurred at 25°C. Near-ultraviolet radiation seemed to have caused higher pycnidium counts than the other light sources.     

Rendiconti e Atti del Congresso Della Societàg Botanica Italiana a Venezia

Abstract

An investigation has been carried out on the effects of some macroelements on the growth and differentiation of carnation meristem-tip. The meristem-tips were cultured over one year period using BAKER and PHILLIPS (1962) modified medium as basal medium. Different concentration of N, P, K and S were used and their effect on the explants was scored. Variation in the K level had no effect on the growth of the meristems except in one case while P at 1/2 X, 1/4 X to 1/8 X levels considerably increased the number of plantlets obtained. N supplied as urea had an inhibiting action on the explant as two different sources of S did.