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     

Comparison of the growth ofCinchona ledgeriana Moens suspension cultures in shake flasks and 7 litre air-lift bioreactors

Summary Suspension cultures ofCinchona ledgeriana Moens have been developed which exhibit good growth in shake flasks with dry weight yields of approximately 9.0 g.l^–1. Cultures have been scaled up for growth in a 7 l air-lift bioreactor. A typical growth curve in the fermenter is shown with similar growth rates but a reduced biomass levels when compared to shake flasks. The analysis of both flask and bioreactor grown suspension cultures indicated the presence of quinidine and low levels of quinine.     

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     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1^–1 4-indole-3yl-butyric acid, 2.0 mg 1^–1 BAP, 0.2 mg 1^–1 gibberellic acid, 50 mg 1^–1 casein hydrolysate and 10 mg 1^–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - GA₃ gibberellic acid - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-but yric acid - IPE initial plating efficiency - NAA 1-naphthaleneacetic acid - f.wt. fresh weight - MES 2-N-morpholinoethane sulfonic acid - MS Murashige and Skoog (1962) - %PE % plating efficiency - PVP-10 polyvinylpyrrolidone (Av. MW 10,000) - FDA fluorescein diacetate     

Influence of operational parameters in the flocculation and production ofLactobacillus plantarum

Summary The influence of different operational parameters, such as the dilution rate (D) and the bleeding rate (B), in the production of a flocculent strain ofLactobacillus plantarum was studied. The effect of the dilution rate was demonstrated to be related to the lactic acid concentration inside the reactor. The effect of the bleeding rate was shown to be critical in the stabilization of the operation (due to a better pH control). It also allowed a continuous recovery of cells outside the reactor. Viability testing of the lactic starter cultures showed that operation with cell purge increased the viability of the starter cultures obtained.Nomenclature B Bleeding rate, h^–1 - D Dilution rate, h^–1 - F Feed flow rate, L h^–1 - I Feed velocity, m h^–1 - Specific growth rate, h^–1 - v Lactic acid specific productivity, g g^–1 h^–1 - P Product concentration (lactic acid), g L^–1 - P _out Product concentration leaving the system, g L^–1 - Q _b Bleeding flow rate, L h^–1 - R Recirculation velocity, m h^–1 - S Substract concentration, g L^–1 - t Time, h - T _p Time of ascensional flow (length of the column/total ascensional velocity), h - T _r Residence time (1/D), h - V Volume of the reactor, L - X Cell concentration, g L^–1 - X _out Cell concentration leaving the system, g L^–1     

Growth ofCatharanthus roseus cell suspensions in bioreactors: On-line analysis of oxygen and carbon dioxide levels in inlet and outlet gas streams

Summary Catharanthus roseus cells (C87N) grown in a 30 litre airlift vessel achieved a growth rate of 0.366 day^–1. The maximum biomass yield (9.13 gl^–1) was recorded after 168 hours (7 days). On-line analysis of the composition of inlet and outlet gas streams during the growth cycle allowed calculation of the metabolic activity of the cultures. Oxygen uptake on a dry weight basis reached a maximum of 4.5×10^–4 Moles O₂ g dry weight^–1 h^–1 after 96 hours (during the mid-logarithmic phase of growth) and a maximum of 2.7×10^–3 Moles O₂ l^–1 h^–1 on a volume basis (towards the end of the logarithmic phase). Carbon dioxide production ran in parallel with oxygen use with maxima at 4.2×10^–4 Moles CO₂ g dry weight^–1 h^–1 and 3.4×10^–3 Moles g l^–1 h^–1 respectively.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. I. Derivatives of L-phenylalanine

Summary Resting cells of a mutant ofArthrobacter sp. (DSM 3747) were used for the bioconversion of D,L-5-benzylhydantoin and related compounds to the corresponding L-amino acids. After optimization of the reaction conditions in shake flask experiments, bioconversions were performed in a preparative scale in a 2-l-bioreactor under nitrogen atmosphere. Specific productivities of 0.4 (p-NO₂-L-phenylalanine) up to 3.9 mM amino acid x g cell dry mass^–1 x h^–1 (p-Cl-L-phenylalanine) were obtained. D,L-5-p-COOH-Benzylhydantoin, D,L-5-phenylhydantoin and D,L-5-p-OH-phenylhydantoin were not accepted as substrates.     

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     

Somatic embryogenesis and regeneration of plants in the bamboo Dendrocalamus strictus

Somatic embryogenesis leading to plant regeneration has been achieved in the bamboo, Dendrocalamus strictus, by culturing seeds (caryopses) on B₅ basal medium supplemented with 2,4-dichlorophenoxyacetic acid. Callus cultures obtained from the embryonal end of the seeds differentiated chlorophyllous embryoids. On transfer to a germination medium (B₅ liquid, sucrose, indolebutyric acid, and -naphthaleneacetic acid) 40% of the embryoids developed into plantlets. Further development of the plantlets occured on B₅ liquid medium (half strength) + sucrose (1%) + IBA (5 × 10^–7M) + NAA (10^–7M).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA -naphthaleneacetic acid     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

Improvement of a mammalian cell culture process by adaptive,model-based dialysis fed-batch cultivation and suppression of apoptosis

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of ~5–7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor ~2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.Abbreviations A membrane area (dm²) - c _Glc,F glucose concentration in nutrient feed (mmol L^–1) - c _Glc,FD glucose concentration in dialysis feed (mmol L^–1) - c _Glc,i glucose concentration in inner reactor chamber (mmol L^–1) - c _Glc,o glucose concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Lac,FD lactate concentration in dialysis feed (mmol L^–1) - c _Lac,i lactate concentration in inner reactor chamber (mmol L^–1) - c _Lac,o lactate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _LS,FD limiting substrate concentration in dialysis feed (mmol L^–1) - c _LS,i limiting substrate concentration in inner reactor chamber (mmol L^–1) - c _LS,o limiting substrate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Mab monoclonal antibody concentration (mg L^–1) - F _D feed rate of dialysis feed (L h^–1) - F _Glc feed rate of nutrient concentrate feed (L h^–1) - K _d maximum death constant (h^–1) - k _d,LS death rate constant for limiting substrate (mmol L^–1) - k _Glc monod kinetic constant for glucose uptake (mmol L^–1) - k _Lac monod kinetic constant for lactate uptake (mmol L^–1) - k _LS monod kinetic constant for limiting substrate uptake (mmol L^–1) - K _Lys cell lysis constant (h^–1) - K _S,Glc monod kinetic constant for glucose (mmol L^–1) - K _S,LS monod kinetic constant for limiting substrate (mmol L^–1) - µ cell-specific growth rate (h^–1) - µ _d cell-specific death rate (h^–1) - µ _d,min minimum cell-specific death rate (h^–1) - µ _max maximum cell-specific growth rate (h^–1) - P _Glc membrane permeation coefficient for glucose (dm h^–1) - P _Lac membrane permeation coefficient for lactate (dm h^–1) - P _LS membrane permeation coefficient for limiting substrate (dm h^–1) - q _Glc cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Glc,max maximum cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Lac cell-specific lactate uptake/production rate (mmol cell^–1 h^–1) - q _Lac,max maximum cell-specific lactate uptake rate (mmol cell^–1 h^–1) - q _LS cell-specific limiting substrate uptake rate (mmol cell^–1 h^–1) - q _LS,max maximum cell-specific limiting substrate uptake rate (mmol cell ^–1 h^–1) - q _Mab cell-specific antibody production rate (mg cell^–1 h^–1) - q _MAb,max maximum cell-specific antibody production rate (mg cell^–1 h^–1) - t time (h) - V _i volume of inner reactor chamber (culture chamber) (L) - V _o volume of outer reactor chamber (dialysis chamber) (L) - X _t total cell concentration (cells L^–1) - X viable cell concentration (cells L^–1) - Y _Lac/Glc kinetic production constant (stoichiometric ratio of lactate production and glucose uptake) (–)     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Ethrel 2-chlorethylphosphonic acid - NAA 1-naphthaleneacetic acid - NR^– nitrate reductase deficient     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

In vitro microrhizome production in Zingiber officinale Rosc.

Microrhizomes of Zingiber officinale were successfully produced from tissue culture derived shoots by transferring them to liquid MS medium supplemented with 1 mg/l BAP, 2 mg/l calcium pantothenate, 0.2 mg/l GA₃ and 0.05 mg/l NAA for shoot proliferation. After 4 weeks of incubation, the medium was replaced with microrhizome induction medium, consisting of MS salts supplemented with 8 mg/l BAP and 75 g/l sucrose. Microrhizome formation started after 20 d of incubation in stationary cultures at 25+1 ° in the dark. Microrhizomes with 1–4 buds and weighing 73.8 to 459 mg each were harvested after 50–60 d. After storage for 2 months in moist sand at room temperature, 80% of the microrhizomes sprouted producing roots and shoots.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Limitations in substrate utilization efficiency by Zymomonas mobilis

Summary Optimal growth conditions for Zymomonas mobilis have been established using continuous cultivation methods. Optimal substrate utilization efficiency occurs with 2.5 g l^–1 yeast extract, 2.0 g l^–1 ammonium sulfate and 6.0 g l^–1 magnesium sulfate in the media. Catabolic activity is at its maximum with glucose uptake rates of 16–18 g l^–1 h^–1 and ethanol production rates of 8–9 g l^–1 h^–1, Q_g values of 22–26 and Q_p values between 11 and 13, which results in 40 g l^–1 h^–1 ethanol yields using a 100 g l^–1 substrate feed. Any increase in these parameters goes on cost of substrate utilization efficiency. Calcium pantothenate can not substitute yeast extract.Abbreviations G Glucose (%) - Pant Calcium pantothenate (mg l^–1) - D Dilution rate (h^–1) - NH₄ Ammonium sulfate (%) - M_g Magnesium sulfate (%) - S₁ Residual glucose in the fermenter (g l^–1) - S₀ Glucose feed (g l^–1) - Eth Ethanol concentration (g l^–1) - GUR Glucose uptake rate (g l^–1 h^–1) - Q_g Specific glucose uptake rate (g g^–1 h^–1) - Q_p Specific ethanol production rate (g g^–1 h^–1) - EPR Ethanol production rate (g l^–1 h^–1) - Y_g Yield coefficient for glucose (g g^–1) - Y_p Conversion efficiency (%) - C Biomass concentration (g l^–1) Present address: (Until June 1982) Institut für Mikrobiologie, TH Darmstadt, 6100 Darmstdt, Federal Republic of Germany