Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Photoautotrophic growth of soybean cells in suspension culture III. Characterization of carbon fixation products under high and low CO2 levels

A photoautotrophic soybean suspension culture (SB-P) was used to study CO₂ assimilation while exposed to elevated or ambient CO₂ levels. These studies showed that under elevated CO₂ (5% v/v) malate is the dominant fixation product, strongly suggesting that phosphoenolpyruvate carboxylase (PEPCase) is the primary enzyme involved in carbon fixation in these cells under their normal growth conditions. Citrate and [aspartate + glutamate] were also significant fixation products during fifteen minutes of exposure to ¹⁴CO₂. During the ten minute unlabeled CO₂ chase however, ¹⁴C-malate continued to increase while citrate and [aspartate + glutamate] declined. Fixation of ¹⁴CO₂ under ambient CO₂ levels (0.037%) showed a very different product pattern as 3-phosphoglycerate was very high in the first one to two minutes followed by increases in [serine + glycine] and [aspartate + glutamate]. Hexose phosphates were also quite high initially but then declined relatively rapidly. Thus, the carbon fixation pattern at ambient CO₂ levels resembles somewhat that seen in C3 leaf cells while that seen at elevated CO₂ levels more closely resembles that of a C₄ plant. The initial fixation product of C₃ plants, 3-PGA, was never detectable under high CO₂ conditions. These data suggest that an in vitro photoautotrophic system would be suitable for studying carbon fixation physiology during photosynthetic and non-photosynthetic growth.Abbreviations SB-P photoautotrophic soybean cells - PEPCase phosphoenol-pyruvate carboxylase - RuBPCase ribulose bisphosphate carboxylase/oxygenase - 3-PGA 3-phosphoglycerate     

Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison

The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (–Fe). Values for K_m and V_max, determined from a Lineweaver-Burk plot, were 57 M and nmoles mg^-1 dry weight for the +Fe cells and 50 M and 22 nmoles mg^-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The K_m and V_max values for Fe-deficient roots were 45 M and 20 nmoles mg^-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.     

Photoautotrophic growth in suspension culture of cells from the moss, Barbula unguiculata

Chlorophyllous cells in suspension culture from the moss, Barbula unguculata Hedw., grown under photoheterotrophic conditions were transferred to photoautotrophic conditions. The cells started to grow photoautotrophically without selection. Maximum growth was observed under irradiances of more than 5 W m² and in an atmosphere enriched with 1% (v/v) CO₂. Under optimum growth conditions, dry weight and chlorophyll content in the culture had increased 20-fold after 20 days of cell growth. High concentration of chlorophyll [10–20 μg (mg dry weight)⁻¹] and high photosynthetic actively [30–70 μmol O₂ evolved (mg chlorophyll)⁻¹ h⁻¹] were observed throughout the culture period. In sugar-free culture medium, cell growth did not occur in the dark or in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under light, although cell growth was observed in glucose-containing medium under those conditions. When cells that were grown photoautotrophically for one year were transferred to glucose-containing medium under ordinary air, they started to grow heterotrophically both in the light and in the dark.     

Establishment and growth characterization of suspension culture of cells from the moss, Sphagnum imbrication

A green-pigmented callus of the moss, Sphagnum imbricatum Hornsch. ex. Russ., was induced and a chlorophyllous cell suspension culture was established using a modified Murashige and Skoog's medium without plant hormones. Cell growth in the light in the presence of glucose started after a short lag and was exponential for 12 days. The chlorophyll level was about 15 μg (mg cell dry weight)⁻¹ and photosynthetic activity ca 20 to 50 μmol O₂ (mg chlorophyll)⁻¹h⁻¹. Cell growth in the light was negligible in the absence of glucose under ordinary air, but photoautotrophic growth was possible under elevated CO₂ concentrations. In the dark, the moss cells grew heterotrophically and continued to synthesize chlorophyll, although at a much reduced rate. The suspension-cultured cells redifferentiated protonemata and shoots when transferred to solid Knop's medium. In contrast to the callus cells, which could not assimilate nitrate, redifferentiated plantlets could use nitrate as the sole nitrogen source.     

Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Photoautotrophic culture conditions and photosynthetic photon flux influence growth of <Emphasis Type="Italic">Lilium</Emphasis> bulblets <Emphasis Type="Italic">in vitro</Emphasis>

Summary Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO₂-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO₂ conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet growth and net photosynthetic rate (NPR) were analyzed. CO₂₋ and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO₂₋ and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic hybrid lilies in vitro     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Photosynthetic properties of two different soybean suspension cultures

The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration.     

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20 g l^-1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO₂ nonenriched (350–450 mol mol^-1 in the culture room) and CO₂ enriched (2,000 mol mol^-1 during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200 mol m^-2 s^-1. The CO₂ concentration in the vessels decreased to approximately 200 mol mol^-1 during the photoperiod on day 21 under CO₂ nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO₄ ^3-, NO₃ ^-, Ca²⁺, Mg²⁺ and K⁺ on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO₂ enriched conditions. The residual percent of PO₄ ^3- was 3% on day 21 under photoautotrophic and CO₂ enriched conditions.Abbreviations MS Murashige & Skoog (1962) basal medium composition - NPR net photosynthetic rate - PPF photosynthetic photon flux     

Relationship between growth of parsley and soybean cells in suspension cultures and changes in the conductivity of the culture medium

Summary Changes in conductivity and pH during the growth cycle of cell suspensions derived from parsley (Petroselinum hortense) and soybean (Glycine max) have been investigated. Measurement of the conductivity of the medium represents a simple, rapid, and reliable method for the precise determination of the growth phase of a culture. The accuracy of this method has been tested by using phenylalanine ammonia-lyase, an enzyme that has a characteristically short, distinct period of activity during the growth cycle of soybean cell suspensions. It is suggested that an automatic regulation of the conductivity of the medium might be employed for growing plant cells in a continuous culture at a defined stage of growth.     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Optimization of culture medium for growth of Haematococcus pluvialis

A central composite rotatable design was used to examine the effects of five components of the medium on the growth of Haematococcus pluvialis in batch culture. The medium components considered were: sodium acetate,potassium nitrate, major elements, trace elements and vitamins. Within the range of the concentrations tested, a moderate concentration of the major elements significantly enhanced algal growth, both in terms of specific growth rate and cell dry weight, whereas the vitamins had no significant effect. Based on the response surface contour plots and the results of numerical analyses, the optimal nutrient concentrations for growth in terms of specific growth rate were 0.51 g L^-1 sodium acetate, 0.25 g L^-1 potassium nitrate, 0.63 mL L^-1 of the major element stock solution and 0.2 mL L^-1 of the trace element stock solution. The optimal nutrient concentrations for biomass production were 1.64 g L^-1 sodium acetate, 0.37 g L^-1potassium nitrate, 2.52 mL L^-1 of the major element stock solution and 0.03 mL L^-1 of the trace element stock solution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of an embryogenic suspension culture of soybean (Glycine max Merrill.)

A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1^–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration.     

A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium

The steady-state metabolic parameters for a murine hybridoma cell line have been determined in continuous suspension culture over a wide range of dilution rates. Long-term adaption occurred over seven months in culture and resulted in lower glucose consumption rates, reduced lactate production, higher cell viability, and, consequently, growth rates more nearly matching the dilution rate. Antibody production rates decreased over the first two months and then remained stable for at least 75 days. The antibody production rate was not found to be growth associated. Steadystate amino acid uptake rates are presented for a wide range of growth rates.