Effect of sodium chloride on growth of heterotrophic marine bacteria

The effect of NaCl on the growth rates and yields of 31 gram-negative, heterotrophic, marine bacteria was determined. The strains used were representative of aerobic genera (Alteromonas, Pseudomonas, Alcaligenes, Bdellovibrio) as well as genera comprised of facultative anaerobes (Beneckea, Photobacterium). Two media were used-the first, a medium designed for the cultivation of marine bacteria and, the second, a medium used for the cultivation of terrestrial strains. These two media differed in the concentrations of divalent cations; the terrestrial medium (TM) contained 2 mM Mg⁺⁺ and 0.55 mM Ca⁺⁺ while the marine medium (MM) contained 50 mM Mg⁺⁺ and 10 mM Ca⁺⁺. The amount of NaCl necessary for optimal growth varied in different strains and was usually considerably higher in TM (100 to 460 mM) than in MM (70 to 300 mM). Many strains which grew in MM and TM had a shorter generation time in the former than in the latter medium. In addition, four strains which grew well in MM usually failed to grow in TM. These results show that higher levels of divalent cations are either essential for growth or stimulate growth rate, indicating that for many marine strains a terrestrial medium modified by the addition of NaCl cannot support optimal growth. Fourteen terrestrial strains of the genera Pseudomonas, Alcaligenes, Acinetobacter, Salmonella, Aeromonas, and Vibrio did not have ionic requirements comparable to those of the marine strains. All of the terrestrial organisms grew in TM without added NaCl (0.068 mM Na⁺ was present as a contaminant). In some terrestrial organisms, growth was stimulated by the addition of NaCl, the highest stimulation being found in Vibrio cholerae. The optimal growth rates and yields for four strains of this species were observed at 2.5 to 5.0 mM NaCl while the growth rates and yields in TM with no added NaCl were 40 to 50% of the optimum.     

Effect of Urea Concentration on Growth of Ureaplasma urealyticum (T-Strain Mycoplasma)

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 × 10⁴ colony-forming units per ml. The maximum yield of organisms, 8.0 × 10⁷ colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 × 10¹⁰ total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K_s value for growth was 2.0 × 10⁻⁴ M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.     

Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulation

In the marine fish intestine luminal, HCO₃ ^? can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO₃ ^? secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH?CStat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10???M FK?+?500???M IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO₃ ^? results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200???M). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen.     

Photosynthetic activity and leaf antioxidative responses of Atriplex portulacoides subjected to extreme salinity

Responses of Atriplex portulacoides upon 40-day-long exposure to salinity (0?C1,000?mM NaCl) were investigated. Mother plants originated from a sabkha located in a semi-arid region of Tunisia. The plant relative growth rate and leaf expansion increased significantly at 200?mM NaCl but decreased at higher salinities. Interestingly, the plants survived salinity as high as 1,000?mM NaCl without displaying salt-induced toxicity symptoms. Despite significant increase in leaf Na⁺ and Cl^? concentrations upon salt treatment, no significant effect on leaf relative water content was registered. Chlorophyll contents and the gas exchange parameters showed a significant stimulation at the optimal salinity (200?mM NaCl) followed by a decline at higher salinities. Extreme salinity hardly impacted the maximal efficiency of photosystem II photochemistry (F _v/F _m), but a marked decrease in the relative quantum yield of photosystem II (??_PSII) was observed, along with a significant increase in non-photochemical quenching (NPQ). Leaf malondialdehyde and carotenoid contents were generally unaffected following salt exposure, whereas those of anthocyanins, polyphenols, and proline increased significantly, being maximal at 1,000?mM NaCl. Leaf superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase (EC 1.6.4.2) activities were significantly stimulated by salinity, whereas catalase (EC 1.11.1.6) activity was maximal in the 0?C400?mM NaCl range. As a whole, protecting the photosynthetic machinery from salt-induced photodamage together with the sustained antioxidant activity may account for the performance of A. portulacoides under high salinity.     

HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROM MODERATELY ACIDIC,OLIGOTROPHIC WATERS 1

Five hydrogen ion buffers were compared for their usefulness in regulating pH in a model oligotrophic, moderately acidic (pH 6.0) algal growth medium. These were 3,3-dimethylglutaric acid (DMGA), tricarbaliylic acid (TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2-(N-morpholino) ethanesulfonic acid (MES). All buffers (2.5 mM) except HEPES limited the reduction of pH in a NH₄⁺-based medium during growth of Chrysochromulina breviturrita Nich. to less than 0.12 units, compared with more than 2 units in an unbuffered medium. Long term growth of C. breviturrita in these media was significantly inhibited (P < 0.05) by TCA and tAA. MES was able to control pH with the minimum amount of NaOH (1.0 mM) added to the medium to adjust to pH 6.0. Four of five bacterial isolates were capable of utilizing tAA as a sole organic-C source, and no isolate could metabolize HEPES or MES. No significant differences (P > 0.05) were found in the maximum growth rates of six algal species (from five classes) in a medium with or without MES buffer, although significantly greater cell yields of Ochromonas danica Prings. were obtained in the buffered medium. MES (pK₄=6.15) was considered to be the most useful buffer in the pH range 5.0–6.5, due to its biological inertness, buffering capacity, the minimal requirement for excess base to adjust pH and its minimal metal complexing ability.     

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3^rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N₂. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg ^?1 DNA with impurities 5.67 and 5.87 μg mg^?1 through buffer I, while 45.77 and 25.53 μg mg^?1 DNA with 6.13 and 6.16 μg mg^?1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C ₁ gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.     

Enhanced autotrophic growth of <Emphasis Type="Italic">Nannochloris</Emphasis> sp. with trona buffer for sustainable carbon recycle

In this study, a potential applicability of a modified Shuisheng-4 culture medium with trona buffer for CO₂(g) capture and the resultant CO₂-rich solution was investigated for the autotrophic growth of Nannochloris sp. Trona is an inexpensive naturally-occurring mineral with high solubility in water. Trona solution after absorbing CO₂(g) controlled at pH ~7 ~ 8 contains high dissolved inorganic carbon (DIC) concentration in water applicable to both open and closed systems and the pH is suitable for the growth. High DIC concentration was also found to enhance the autotrophic growth rate when the Na⁺ concentration is ~ < 0.12 M. However, the tolerance of a marine green alga, Nannochloris sp., to Na⁺ ion was found to twice as much as that of a freshwater green alga, Chlorella vulgaris. Therefore, it is anticipated that trona buffer solution has potential for better CO₂ utilization for the mutual benefit of microalgae-derived biofuels production and carbon recycle.     

Manganese dependent production of 5′-nucleotidase by alkalophilic Bacillus No. C-3

Alkalophilic Bacillus no. C-3 isolated from soil produced 5′-nucleotidase (EC 3.1.3.5) extracellularly when cultured in a medium containing Mn²⁺. The unique point of enzyme production is that the enzyme was produced well in the medium containing a rather high concentration of Mn²⁺, in spite of a small difference in growth. The optimum concentration of Mn²⁺ for the enzyme production was 10 mM and over. Mn²⁺ could not be replaced by other divalent cations when added singly. In the presence of 10 mM Mn²⁺, the enzyme production was repressed by the addition of 0.5 mM phosphate to the medium. The course of the enzyme production closely paralleled the increase in growth. The optimum pH for the enzyme activity was 9.2–9.5, and KHCO₃-K₂CO₃ buffer was suitable for the enzyme.     

Nitrogen Metabolism in Soybean Tissue Culture: II. Urea Utilization and Urease Synthesis Require Ni

Potassium citrate (10 mM, pH 6) inhibits the growth of cultured (Glycine max L.) cells when urea is the sole nitrogen source. Ureadependent citrate toxicity is overcome by three separate additions to the growth medium: (a) NH₄Cl (20 mM); (b) high levels of MgCl₂ (10 mM) or CaCl₂ (5-10 mM); (c) low levels of NiSO₄ (10⁻² mM). Additions of 10⁻² mM NiSO₄ not only overcome citrate growth inhibition but the resultant growth is usually better than urea-supported growth in basal medium (neither added citrate nor added nickel). In the absence of added citrate, exceedingly low levels of NiSO₄ (10⁻⁴ mM) strongly stimulate urea-supported growth in suspension cultures.     

Isolation and properties of isocitrate lyase from Lupinus seed

Isocitrate lyase (threo-d_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) was purified from cotyledons of Lupinus seedlings. The final preparation showed two bands after polyacrylamide-gel electrophoresis. The optimum pH using phosphate, Tris or imidazole buffer was at pH 7.5; with triethanolamine (TRA) it was at pH 7. The enzyme required Mg²⁺ for maximal activity, and N-ethylmaleimide (NEM) inactivated the enzyme. Activity was increased by incubation with the reducing agents, glutathione (GSH), acetylcysteine (acetylcys), dithionite (Na₂S₂O₄), thioglycolate (TG) or 1,4-dithioerythritol (DTE). Na₂S₂O₄ and DTE were the most active among the tested substances and DTE prevented much of the inactivation by NEM. The apparent K_m value for isocitrate was ca 1 mM in phosphate buffer at pH 6.8 or 7.5 but was substantially lower (0.1–0.2 mM) using Tris, TRA or imidazole buffers. Glyoxylate, oxalate and malonate were competitive inhibitors of the enzyme. Synthase activity of the enzyme (i.e. formation of isocitrate from succinate and glyoxylate) was demonstrated. The K_m values for glyoxylate and succinate were 0.05 and 0.2 mM, respectively. The addition of glyoxylate to the culture medium in which Lupinus seeds germinate resulted in a reduced development of isocitrate lyase activity during germination.     

Octanoic acid uptake in Pseudomonas putida U

Pseudomonas putida U grown in a chemically defined medium containing octanoic acid as the sole carbon source accumulated a homopolymer of poly(3-hydroxyoctanoate) as intracellular reserve material, and metabolized the polymer during the late exponential phase of growth. Kinetic measurement of the uptake of [1-¹⁴C]octanoic acid by cells at 34°C in 85 mM phosphate buffer, pH 7.0 showed linear uptake for at least 2 min and the calculated K_m and V_max were 100 μM and 9 nmol min⁻¹ respectively. This transport system is constitutive, energy-dependent, and is strongly inhibited by structural analogs of octanoic acid, by various fatty acids with a carbon length higher than C₅ and by certain phenyl derivatives.     

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log₁₀ lower than in ZoBell marine broth 2216. The fates of ¹⁴C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.     

Inducement of a Heat-Shock Requirement for Germination and Production of Increased Heat Resistance in Bacillus fastidiosus Spores by Manganous Ions

Bacillus fastidiosus, which requires uric acid or allantoin, grows and sporulates on a simple medium containing 59.5 mM uric acid, 5.7 mM K₂HPO₄, and 2% agar in distilled water. Seventy to ninety percent sporulation was achieved in 96 h. Spores obtained on this medium do not need a heat shock prior to germination. The necessary germination conditions for this organism are 30 C, phosphate or this(hydroxymethyl)aminomethane buffer at pH 7.0, and 5.95 mM uric acid. Sporulation occurred earlier (48 h) and with higher frequency (greater than 99%) when Mn²⁺ was added to the growth medium. However, these spores germinated only after heat activation (70 C, 30 min). The effectiveness of heat activation was directly dependent upon the concentration of Mn²⁺ in the growth medium; 10⁻⁵ M Mn²⁺ was the minimal concentration for the effect. This phenomenon was not found upon addition of Ca²⁺, Mg²⁺, Fe²⁺, Zn²⁺, or Cu²⁺ to the medium. The Mn²⁺ content of the spores depended upon the concentration of Mn²⁺ in the sporulation medium. There was a significant difference in heat resistance between spores harvested from unsupplemented medium and those harvested from medium supplemented with 5 × 10⁻⁵ M Mn²⁺. A D_{85 C} value of 6.5 min was determined with the former, whereas the latter had a value of 17.0 min. Very little change in either Ca²⁺ or dipicolinic acid content was detected in spores harvested from various Mn²⁺-supplemented media. Thus Mn²⁺ may play a role in the inducement of the heat-shock requirement and the formation of spores with increased heat resistance.     

Non-identity of cystine lyase with β-cystathionase in turnip roots

An active preparation of cystine lyase (EC 4.4.1.-) was prepared from turnip roots and its substrate specificity examined. Only L-cysteine, cysteine-S-SO₃, and the sulphoxides of L-djenkolic acid, S-methyl-and S-ethyl-L-cysteine were substrates. L-Cystathione, L-djenkolic acid, S-methyl-and S-ethyl-cysteines were not cleaved by this enzyme. The K_m for L-cystine was 1.3 mM and L-cystathionine acted as an effective competitive inhibitor with a K_i of 0.7 mM. After dialysis against 10 mM potassium phosphate buffer pH 7.5, added pyridoxal phosphate was absolutely necessary for activity. In addition a marked stimulation was observed in the presence of ammonium sulphate. The products of the reaction were cysteine persulphide, pyruvate and presumably ammonia. The persulphide was easily demonstrated by cleavage with CN^? to yield SCN^? under conditions in which elemental sulphur was unreactive.     

The use of HEPES as a buffer for the growth of the cyanobacterium Anacystis nidulans

Summary Of a number of possible buffers only HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) supported continued and maximum growth of photoautotrophically grown Anacystis nidulans when grown in medium C and aerated with air (0.03% CO₂). Cultures aerated with 1% CO₂ in air had adequate buffering without the use of additional buffers in medium C. With either air or 1% CO₂ grown cells, phosphate and TRICINE (N-tris-hydroxymethyl-methylglycine) buffers gave growth rates of 67% compared to HEPES buffer while complete inhibition of growth occurred with TRIS (tris-hydroxymethylaminomethane) buffer.     

Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.     

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO₃^?) in the growth medium. At NO₃^? concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO₃^?, the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO₃^? uptake at low NO₃^?, while GABA inhibited NO₃^? uptake at high NO₃^?. Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Growth and triterpenic acid accumulation of Cyclocarya paliurus cell suspension cultures

Cyclocarya paliurus is a unique plant growing in central China with hypoglycaemic and hypolipaemia effects. To make better use of this functional food resource, cell suspension cultures and triterpenic acid accumulation were studied. Stable and uniform cell suspension cultures were established in liquid basal Murashige and Skoog medium supplemented with 2,4-dichlorophenoxy acetic acid (0.5 mg/L), naphthalene acetic acid (0.3 mg/L) and cytokinin (1.0 mg/L). According to the growth curve and triterpenic acid accumulation curve, the 8 ～ 10^th day postinoculation was the optimum time for subculture, and the 14^th day was the optimum time for harvest. Murashige and Skoog medium and woody plant medium were suitable for both cell growth and triterpenic acid accumulation. 3% sucrose (w/v), 60 mM total nitrogen (NO₃ ^?/NH₄ ⁺ = 2/1), 1.25 mM KH₂PO₄, 2 mM CaCl₂, and 2 mM MgSO₄ were all found to be fit for cell growth and triterpenic acid accumulation in a cell suspension culture of Cyclocarya paliurus. Total triterpenic acid, ursolic acid and oleanolic acid content in suspended cultured cells were all significantly higher than that of leaves and calluses (P ? 0.01), with levels up to 6.24, 2.28, and 0.94% (of dry weight), respectively. The betulinic acid content of suspended cultured cells also reached 0.82%, which was significantly higher than that of calluses. These results suggest that suspended cultured cells of Cyclocarya paliurus were rich in triterpenic acids and could be used for the production of total triterpenic acid, ursolic acid, oleanolic acid and betulinic acid.