Effect of seaweed extracts on the growth and biochemical constituents of Vigna sinensis

The effect of seaweed liquid fertilizers (SLF) of Sargassum wightii and Caulerpa chemnitzia on growth and biochemical constituents of Vigna sinensis was studied. The seeds soaked with aqueous extract of seaweeds performed better when compared to the water soaked controls. Hundred per cent germination was recorded both in aqueous extract soaked and water soaked treatments. The low concentration (20%) of aqueous extracts of S. wightii and C. chemnitzia promoted the seedling growth including the parameters of shoot length (15.87, 14.13 cm/seedling), root length (6.42, 5.38 cm/seedling), fresh weight (4.017, 4.012 g/seedling) and dry weight (0.878, 0.865 g/seedling), chlorophyll (1.599, 1.491 mg g-1 fr. wt.), carotenoids (0.899, 0.875 mg g-1 fr. wt.), protein content of shoot (3.956, 3.474 mg g-1 fr. wt.) and root (2.926, 2.890 mg g-1 fr. wt.), amino acid content of shoot (1.447, 1.429 mg g-1 fr. wt.) and root (0.698, 0.680 mg g-1 fr. wt.), reducing sugar content of shoot (6.426, 6.233 mg g-1 fr. wt.) and root (5.118, 5.103 mg g-1 fr. wt.), total sugar content of shoot (11.846, 11.350 mg g-1 fr. wt.) and root (10.368, 10.102 mg g-1 fr. wt.), alpha-amylase (1.927, 1.819 microg min-1 mg-1 protein) and beta-amylase (1.730, 1.617 microg min-1 mg-1 protein) activities in V. sinensis. Among the two seaweeds tested, S. wightii exhibited better responses.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

Distribution of naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana: molecular events in prey capture

Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana.     

Ethylene Production by Tobacco (Nicotiana tabacum) Callus

Tobacco callus cultures grown on defined agar-solidified media produced ethylene in differing amounts, which were related to cultural treatment and age of the callus. There was a close correlation between the rate of ethylene production and growth. In darkness, maximal rates occurred in the third week of growth with ethylene production in the range of 750 nl (callus piece)^?1 d^?1 (fr. wt. = 1.5 g), and in the light, maximal rates occurred in the first week of growth, 200 nl (callus piece)^?1 d^?1 (fr. wt. = 200 mg). Growth was also correlated with ethylene production when the latter was altered by exposure of the callus to inhibitors of ethylene synthesis, L-canaline, benzyl isothiocyanate, and 3,5-diiodo-4-hydroxy-benzoic acid. No correlation was found following treatment with AgNO₃, a presumptive inhibitor of ethylene action. The inhibition of growth and ethylene production by L-canaline was partially reversed by gassing the cultures with ethylene (1 μl/1). A mercuric perchlorate sink had no significant effect on growth. A possible relationship between ethylene evolution and growth is discussed.     

Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.)

Buckwheat (Fagopyrum esculentum Moench.) is a potentially important source of rutin, a natural flavonoid with antihyperglycemic, antihypertensive, and antioxidative properties. To examine in vitro production of rutin, we established a hairy root culture of buckwheat by infecting leaf explants with Agrobacterium rhizogenes R1000, and tested the growth conditions and rutin production rates of these cultures. Ten hairy root clones were established; their growth and rutin production rates ranged from 233 to 312 (mg dry wt per 30 mL flask, and 0.8 to 1.2 (mg/g dry wt), respectively. Clone H8, which had high growth and rutin production rates (312 mg dry wt per 30 mL flask and 1.2 mg/g dry wt, respectively), was selected for further experiments. H8 showed maximal growth and rutin content at 30 days in culture in MS medium. Of four tested culture media, half-strength MS medium was found to induce the highest levels of growth (378 mg dry wt per 30 mL flask) and rutin production (1.4 mg/g dry wt) by clone H8. In contrast, supplementation with auxins (0.1-1 mg/l IAA, IBA and NAA) increased the growth rate, but had no significant effect on rutin production by H8. Collectively, these findings indicate that hairy root cultures of buckwheat culture could be a valuable alternative approach for rutin production.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Uptake of phosphate and its effect on phenylalanine ammonia-lyase activity and cinnamoyl putrescine accumulation in cell suspension cultures of Nicotiana tabacum

The influence of phosphate on the medium-induced formation of cinnamoyl putrescines in cell cultures of Nicotiana tabcum was investigated. Phosphate added to a phosphate-free production medium was completely accumulated in the cells within 24h after inoculation at initial concentrations up to 2 mM. At higher concentrations phosphate was partly accumulated with an intracellular saturation at approx. 0.65 mmol/g dr. wt. equivalent to approx. 45 mM intracellular concentration. Enhanced activities of phenylalanine ammonialyase and increased product levels of cinnamoyl putrescines, induced by cell transfer into phosphatefree medium were suppressed similarly at initial phosphate concentrations of 0.02–0.5 mM. At the same time growth was stimulated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - fr. wt. fresh weight - dr. wt. dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 g/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 g morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 g morphine equivalents/g fr. wt.) by ELISA.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - HF phytohormone-free - NAA 1-naphthaleneacetic acid - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography     

Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.     

Effect of culture process on alkaloid production by Catharanthus roseus cells. II. Immobilized cultures.

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10-13 g l-1). The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.     

Studies on the biosynthesis of phenols in fungi. Conversion of [14C]orsellinic acid and [14C]orcinol into fumigatol by Aspergillus fumigatus I.M.I. 89353

1. Sodium [1-(14)C]acetate was incorporated into orsellinic acid and fumigatol by Aspergillus fumigatus. 2. [(14)C]Orsellinic acid was prepared biosynthetically. It was converted almost entirely into fumigatol and fumigatin within 2 days of supplementation of the medium. The apparent decrease in incorporation after a longer period of growth was due to decomposition of radioactive fumigatol and the production of relatively unlabelled material. The addition of orcinol to these cultures decreased the conversion of [(14)C]orsellinic acid into fumigatol. [(14)C]Orsellinic acid was incorporated into 3,4-dihydroxytoluquinol in both sets of cultures. 3. [(14)C]Orcinol was prepared from [(14)C]orsellinic acid after acid hydrolysis. It was also very effective as a precursor of fumigatol (60% incorporation). 4. The specific activity of fumigatin was lower than that of fumigatol at early stages of growth (4-5 days after inoculation) with all the labelled substrates that were tested. This indicated that fumigatin arose from fumigatol after oxidation in the medium. 5. The presence of orcinol in the medium greatly stimulated the incorporation of radioactivity (presumably derived from the (14)CO(2)H of orsellinic acid) into the isoprenoid compounds, ergosterol and ubiquinone, in the mycelium.     

Flower-Inducing Activity of Water Extracts of Various Plant Species, in Particular Pharbitis nil

The crude water extracts of leaves of many plant species belongingto Spermatophyta and some belonging to Bryophyta induced floweringof Lemna paucicostata 151 (PI51) under continuous light, atthe concentrations equivalent to 0.1 to 10 mg fr wt leaf per10 ml culture medium (mg fr wt/10 ml). The extract of Salvinia(Pterydophyta) added together with the extract of Lemna at aconcentration lower than that necessary to cause flowering alsoinduced flowering. The activity of the water extracts of someplants varied considerably from experiment to experiment dueto unknown factors, but the extracts of Pharbitis nil strainViolet, a sensitive short-day plant, always showed a high activity,as did the extracts of Lemna paucicostata reported previously. The extract of Pharbitis cotyledons induced flowering of P151even at 0.3 mg fr wt/10 ml, and significantly promoted floweringof L. paucicostata 441 and 6746 at 1–3 mg fr wt/10 ml.Ex-udate from the cuttings of the seedlings was also active.However, neither the activity of the water extract nor thatof the exudate could be correlated with photoperiodic floralinduction. On the other hand, the extract of leaves or cotyledonshad higher activity (on a fr wt basis) than that of other organs,and the activity of the extract of cotyledons changed with theirage roughly in parallel with their photoperiodic sensitivity. (Received April 17, 1989; Accepted August 10, 1989)     

High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system

Thraustochytrids, in particular Schizochytrium spp., are used for the production of the valuable polyunsaturated fatty acid, docosahexaenoic acid (DHA; 22:6 n-3). Growth of Schizochytrium sp. G13/2S in a defined medium was initially made in shake-flask cultures to determine the optimum concentrations of glucose (100-200 g l(-1)) and ammonia ( approximately 300 mg l(-1)) that could be used by this microorganism. In subsequent fermenter cultures, a pH-auxostat method was used to maintain NH(3) from 200-300 mg l(-1). During the first 49 h of fermentation, 150 g glucose l(-1) produced 63 g cell dry wt l(-1). Although growth was not limited by the supply of nitrogen, total fatty acids were at 25% cell dry wt which is more than half the final lipid content of commercially-grown Schizochytrium biomass which uses N-limited medium in the final stages for maximum lipid accumulation. This strategy is therefore useful for the cultivation of Schizochytrium to a high cell density up to the point when lipid accumulation can be triggered by N exhaustion.     

Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili

The influence of osmotic stress on capsaicin production was investigated in cell suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili, a chili species native to Northeastern India. The sterilized seeds were germinated in Murashige and Skoog medium. Two-week-old hypocotyls were excised from in vitro germinated seedlings and implanted in MS medium containing 2, 4-dichlorophenoxyacetic acid (2?mg/l), and Kinetin (0.5?mg/l) for callus induction. Capsaicin production in the suspension cultures was significantly affected using sucrose, mannitol, and NaCl in the medium. Stoichiometric analysis with different combinations of sucrose and non-sugar osmotic agent (NaCl) showed that osmotic stress was an important factor for enhancing capsaicin production in cell suspension cultures of C. chinense. The capsaicin content of 1,644.1???g?g^?1 f.wt was recorded on day 15 in cultures grown in MS medium containing 87.64?mM sucrose in combination with 40?mM NaCl. However, osmotic stress treatment at 160?mM NaCl with sucrose resulted in lowering capsaicin accumulation and separation of cell wall from their cytoplasm, under microscopic observation.     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Diurnal Extension Rates of Wheat Leaves in Relation to Temperatures and Carbohydrate Concentrations of the Extension Zone

The objective of this experiment was to determine how diurnalvariations in rates of leaf extension of wheat plants in anirrigated field crop were related to temperatures and carbohydrateconcentrations of the extension zone. Leaves 3, 4. 5 of themain shoot were studied as each emerged from the encirclingsheath. The carbohydrates in the extension zone of the emergingleaf were analysed by converting them to glucose-6-phosphateand then measuring the reduction of NADP in the presence ofglucose-6-phosphate dehydrogenase. Average hexose concentrations(glucose and fructose) increased each day from 4 up to 5 mgg^–1 fr. wt. and sucrose from 3 up to 7 mg g^–1 fr.wt. with the maximum in mid-afternoon; there were no differencesamong the three leaves. Concentrations of fructans were constantthroughout the day for leaves 3 and 4 but showed a mid-afternoonrise in leaf 5. The average concentrations of fructans in theextension zones increased from 0 to 5 to 11 mg g^–1 fr.wt. for leaves 3, 4, and 5 respectively and was consistent withthe conclusion that there was an increasing over-supply of carbohydratesfor growth of the shoot as the plant increased in size. Ratesof leaf extension were correlated with temperature but not withhexose concentrations. We concluded that the supply of carbohydratesdid not limit the growth of leaves under field conditions buttheir utilization in leaf growth was limited by temperature.The rates of leaf extension increased exponentially with temperatureand the relationship was described by the Arrhenius equation.The Q10 at 15 °C for leaf extension was 2.7 for leaves 4and 5 and 3.2 for leaf 3.     

Improvement of the catharanthine productivity in hairy root cultures ofCatharanthus roseus by using monosaccharides as a carbon source

Summary Sucrose, glucose, and fructose as carbon sources in culture medium were assessed in hairy root cultures ofCatharanthus roseus. The cultures preferentially consumed sucrose, resulting in about 40% (dry wt) higher growth rate. However, fructose enhanced the cathranthine yield about two-fold. The elevated yield was not seemingly ascribed to the higher osmolarity per unit weight of fructose than sucrose. A two stage culture using sucrose (1st) and fructose (2nd) improved volumetric yields of catharanthine about two-fold, i.e. 41 mg/l.     

Effects of precursors on serially propagated Digitalis lanata leaf and root cultures

Serially propagated Digitalis lanata leaf and root cultures established from germinated seeds were studied for digoxin production. Leaf cultures were grown and maintained in a medium containing benzyl adenine, and root cultures in the same medium with indoleacetic acid. A consistently high digoxin content, as determined by radio-immunoassay, is present in the leaf culture (9.0 mg % dry wt) and in root culture (1.9 mg % dry wt) as compared to unorganized cells (0.06 mg % dry wt). Leaf liquid cultures grew very rapidly as compared to the root cultures and the unorganized cell suspension cultures. The concentration of digoxin increased in both leaf and root cultures by adding to the medium either sodium glycocholate, cholesteryl acetate, or progesterone. Smilageninacetate increased the digoxin content of root cultures but not that of leaf cultures. Lanosterol and 5β-androstan-3,17-dione did not significantly increase the concentration of digoxin. Deoxycholic acid was toxic to the tissues studied.