Production of the triterpenoid quassin in callus and cell suspension cultures of Picrasma quassioides Bennett

Plant cell and suspension cultures have been established from stem cuttings of Picrasma quassioides Bennett. The effect of 244 different types/concentrations of plant growth regulators on growth and quassin accumulation in callus tissue was investigated. Best growth, in terms of wet/dry weight after four weeks growth, was obtained on B5 media supplemented with 2% glucose, 10% coconut milk, 0.5 mg.l^–1 zeatin riboside and 1.5 mg.l^–1 IBA. The highest yields of quassin (0.014–0.018%) were detected on this same media supplemented with 1.0 mg.l^–1 IBA and varying concentrations of zeatin riboside. Suspension cultures were easily established on B5 media supplemented with 2% glucose, 1.0 mg.l^–1 2,4-D and 0.5 mg.l^–1 kinetin. The carbon source had a marked effect on quassin accumulation with 0.32% quassin being detected when cells were grown in 2% galactose. This is comparable to the highest reported quassin yield for the whole plant.Abbreviations IAA indole-3-acetic acid - IBA indolebutyric acid - IpA N⁶-(-isopentenyl) adenine - IpAR N-(-isopentenyl) adenine riboside - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6BA 6-benzyladenine     

Growth rate and methane affinity of a turbidostatic and oxystatic continuous culture ofMethylococcus capsulatus (Bath)

Summary A turbidostatic and oxystatic fermentation system was used to study the growth kinetic ofMethylococcus capsulatus (Bath). Dissolved oxygen and methane concentrations were measured continuously with membrane inlet mass spectrometry. The specific growth rate was found to increase from 0.25 h^–1 to 0.37 h^–1 and the saturation constant for methane was found to decrease from 71 M to 1.3 M as the copper content of the medium was varied from a very low to a high value.     

Production of pullulan by a fed-batch fermentation

Summary In pullulan production from sucrose byAureobasidium pullulans, a sugar concentration higher than 5% (w/v) inhibited cell growth and the production of exopolysaccharide. By a fed-batch fermentation, the inhibitory effects of the high sugar concentration were overcome and 58.0 g/1 of exopolysaccharide were obtained from 10% sucrose.Abbreviations m, n relationship parameters for the growth and non-growth associated product formation - X, Xmax biomass and maximum biomass concentration (g cell/1) - P product concentration (g exopolysaccharide/1) - specific growth rate of cell (hr^–1)     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos

Organogenesis and somatic embryogenesis were induced from megagametophyte and zygotic embryo explants of 2 cycad species, Ceratozamia hildae and C. mexicana, cultured on modified B5 medium containing kinetin (0–13.9 M) and 2,4-d (0–9.0 M). Organogenesis occurred from megagametophyte explants of both species on the range of media tested. Somatic embryogenesis was largely restricted to zygotic embryo explants. Somatic embryos germinated in vitro: however, rooting of adventitious shoots was unsuccessful.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid R-014515.     

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L^–1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L^–1 NAA and 1 mg·L^–1 K (N₂K₁MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d^–1 and 0.08 d^–1 were obtained in MS medium supplemented with 1 mg·L^–1 NAA, 0.1 mg·L^–1 K and 30 g·L^–1 sucrose (N₁K_0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L^–1 sucrose (N₁K_0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N₁K_0.1MS and N₁K_0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L^–1 and 7.9 g·L^–1, and yields on carbohydrate were 0.39 and 0.45 in N₁K_0.1MS and N₁K_0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.Abbreviations B5 Gamborg mineral salt medium - d.w. Dry weight - K Kinetin - MS Murashige and Skoog mineral salt medium - N or NAA Naphthaleneacetic acid - N_iK_jMS i and j are the respective concentrations (mg·L^–1) of NAA and K - n Number of experimental points - r Linear regression correlation coefficient     

Growth,nitrogen fixation,and mass culture of isolatedAnabaena azollae

Summary An independent strain ofAnabaena azollae was evaluated for its potential as a biofertilizer in wetland rice fields. Sustained rapid growth (doubling time=10.5 h) and nitrogenase activity (32 nmol C₂H₄ h^–1 g^–1 chl) was recorded. Mass cultivation (up to 300 litres) for the first time with this species was also achieved.     

Linear growth in microbial cultures limited by accumulated substrate

Summary The linear growth phase in cultures limited by intracellular (conservative) substrate is represented by a flat exponential curve. Within the range of experimental errors, the presented model fits well the data from both batch and continuous cultures ofEscherichia coli, whose growth is limited in that way.List of symbols D dilution rate, h^–1 - K_S saturation constant, g.L^–1 - S concentration of the limiting substrate, g.L^–1 - S_i concentration of the limiting substrate accumulated in the cells, g.g^–1 - S_o initial concentration of the limiting substrate, g.L^–1 - t time of cultivation, h - t₁ time of exhaustion of the limiting substrate from medium, h - t_o beginning of exponential phase, h - X biomass concentration, g.L^–1 - X₁ biomass concentration at the time of exhaustion of the limiting substrate from the medium, g.L^–1 - X_o biomass concn. at the beginning of exponential phase, g.L^–1 - biomass concn. at steady-state, g.L^–1 - Y growth yield coefficient (biomass/substrate) - specific growth rate, h^–1 - _m maximum specific growth rate, h^–1     

Cell aggregates in wheat suspension cultures and their effects on isolation and culture of protoplasts

Fine embryogenic suspension cultures of wheat (Triticum aestivum cv Hartog and Timmo, and T. durum cv D6962) tend to grow into large cell clumps (1–3 mm), resulting in the formation of mixed suspension cultures consisting of both fine and large cell clumps. The cell clumps were separated according to their sizes and cultured as new lines to investigate their growth rate and differentiation potential and the effects of cell aggregate size on protoplast culture. The results showed that the fine clusters (<310 m) had a higher growth rate but a lower differentiation frequency than the large cell aggregates (310–2000 m). After 2–4 weeks incubation, all the new lines reformed mixed suspension cultures again. The large clumps (>1100 m) released fine cell clusters into the medium so it was possible to initiate fine embryogenic suspension cultures from the large clumps. With regard to the isolation and culture of protoplasts, although the highest yield of protoplasts was obtained from the fine cell clusters, the protoplasts isolated from different sized cell aggregates all had similar potential for sustained cell division and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation

Protoplasts of the nicotinamide-deficient Hyoscyamus muticus cell line nic^– IVH2 and of the nitrate reductase-deficient Nicotiana tabacum cell line NR^– cnx 68 were induced to fuse. Selection for putative interspecific hybrid clones was via auxotroph complementation. Controls included tests for cross-feeding, cross inhibition, PEG-induced variation, culture-induced variation, reversion, viability, delayed selection. Protrotrophic cell lines were recovered exclusively from PEG-treated mixtures of both protoplast types. The putative hybrid clones grew independent of exogenously supplied auxins and cytokinins, and at a faster rate than either parent. The morphogenic potential of different clones varied from non-morphogenic to regeneration of fertile plants. Indirect evidence for the hybrid nature of the clones is provided from a) tight selection, b) hormone-independent growth, c) hybrid vigour, d) extreme morphological variation, e) isoenzyme bands from both parents, f) morphogenic potential. Definite proof for the hybrid nature was, however, provided from species-specific DNA hybridization. Chimerism could be excluded since only the large subunit of Hyoscyamus muticus ribulose bisphosphate carboxylase was found and since species-specific DNA hybridization identified clones which gave no Nicotiana tabacum signal.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BAP 6-Benzylaminopurine - PEG Polyethyleneglykol - MES (2[N-morpholino]ethane sulfonic acid - Tris-HCl Tris(hydroxymethyl)-aminoethane-HCl % is given throughout in w/v - FMI Friedrich Miescher-Institut     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Plant regeneration from embryogenic suspension cultures of oil palm (Elaeis guineensis Jacq.)

Suspension cultures of oil palm (Elaeis guineensis Jacq.) were established in a medium with 80 or 100 mg.l^–1 2,4-dichlorophenoxyacetic acid and 1 g.l^–1 activated charcoal, from calli producing embryogenic cells and protruding proembryos. The suspension was composed of meristematic clumps, breaking away and giving rise to new smaller aggregates. Under the best conditions, the initial weight increased about 4 fold in one month. Embryo differentiation was achieved when plating the clumps either directly, or after a phase in hormone-free liquid medium. In the second case, early maturation occurred in liquid medium. Secondary embryogenesis was reduced, and up to 18.1 % embryos formed shoots. Rooted plants were sucessfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Protoplast culture and plant regeneration ofPinellia ternata

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10⁵ protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1^–1) and NAA (0.2 mg 1)^–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KT kinetin - CH casein hydrolysate