High density cultivation of plant cells in a new aeration-agitation type fermentor,maxblend fermentor®

The applicability of a new aeration-agitation type fermentor with a grid-paddle type impeller and a spiral-sparger, Maxblend Fermentor® (MBF) for high density cultivation of plant cells, was investigated. The MBF showed a high capacity for oxygen supply and extremely low hydrodynamic stress in aeration and mixing compared with a conventional fermentor (CF). When Oryza sativa cells were cultivated at a k_La of 20 h⁻¹, a high cell density cultivation of about 30 g dry cell weight per liter was accomplished in both fermentors and there were few differences in culture performance between the two. On the contrary, considerable differences were observed when Catharanthus roseus cells, which seemed to be sensitive to physical stress, were cultivated at a k_La of 20 h⁻¹ in both fermentors. The MBF exhibited excellent cell growth characteristics, achieving about 19 g dry cell weight per liter, because of its superior oxygen supply and low hydrodynamic stress in aeration and mixing in highly viscous cultures containing high density cells. In CF only about 9.5 g dry cell weight per liter was achieved because of its high hydrodynamic stress.     

Development of an optical method for monitoring protoplast formation from cultured plant cells

The size distribution of cell aggregates during protoplast isolation from Catharanthus roseus and Nicotiana tabacum was measured by a Coulter counter. It was observed that a gradual reduction in the size of cell aggregates occured during protoplast formation. A previously developed specialized spectrophotometer for the photometric measurement of plant cell concentration was used for continuous monitoring of the reduction in the size distribution of cell aggregates during protoplast formation. This made it possible to use changes in optical density (O.D.) to distinguish the three stages in protoplast formation—plasmolysis, maceration and cell wall digestion. During the processes of maceration and cell wall digestion, the O.D. decreased and reached a steady value at the end of each process. Consequently, changes in the O.D. could be used to determine precisely the end of each process. The cell wall digestion process was described by a simple first order reaction model and the rate of protoplast formation (cell wall digestion) was quantitatively evaluated from the rate constant (k) of this reaction. By using the values of k, the optimal enzymatic reaction conditions for isolating protoplasts from C. roseus and N. tabacum cells were determined.     

Distribution of alginate oligomers in Catharanthus roseus and Wasabia japonica cells cultured in a medium containing alginate oligomers

Distribution of alginate oligomers (AO) which are endogenous elicitor-like substances, in cultured plant cells were investigated by using AO conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (ANDS). When AO-ANDS was added at 0.5 g l^–1 to the Catharanthus roseus cell culture, it adhered to the cells as observed by fluorescence microscopy. Using protoplasts of C. roseus, AO-ANDS was found not only in the cell walls but also in the cell membrane and cytoplasm. When C. roseus was cultivated in a medium containing oligo-galacturonic acids, as an endogenous elicitor, this was also found in the cell wall, cell membrane and cytoplasm of C. roseus cells. Similar results were also obtained with Wasabia japonica cells.     

Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R ²>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.     

Opposite effects of synthetic auxins, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid on growth of true ginseng cell culture and synthesis of ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 10⁴ μm³ were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 10⁴ μm³, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.     

High-level production of arbutin from hydroquinone in suspension cultures of Catharanthus roseus plant cells

Summary Plant cell suspensions of Catharanthus roseus efficiently converted exogenously supplied hydroquinone (HQ) into arbutin. Arbutin productivity of the cells was strongly influenced by the growth stage of the cultivated cells and by the manner of the addition of HQ. We have developed two methods: (i) cultivating suitable cells for producing arbutin at high density; (ii) efficiently adding toxic HQ to the cells. The yield of arbutin could be increased up to 9.2 g/l (45% of cell dry weight), which is the highest yield in the field of plant biotechnology. Repeated examinations and scaling up to a 20-l jar fermentor suggested that C. roseus cells stably produce arbutin in large amounts under the established conditions. Offprint requests to: S. Inomata     

Caffeic acid-O-methyltransferase in a suspension of cell aggregates of tobacco

Aggregates of tobacco cells in suspension in 2,4-D (10^?6 M) and kinetin (10^?5 M) cultures were fractionated by size, then their O-methyltransferase (OMT) activities were assayed. Only the kinetin culture showed high OMT activity, which was higher in the larger than the smaller aggregates at all stages of cell growth. The contents of phenolic acids were also greater in the larger cell aggregates in the kinetin culture. However, when the kinetin cultured cells were transferred to a medium containing 10^?6 M of 2,4-D, the relationships between the cell size of the aggregates and OMT, lignin and the phenolic acids disappeared. The importance of kinetin and cell association for OMT and the subsequent lignification of the cells is discussed.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

ACC deaminase containing diazotrophic endophytic bacteria ameliorate salt stress in Catharanthus roseus through reduced ethylene levels and induction of antioxidative defense systems

Among a total of 27 cultivable salt tolerant endophytic bacteria isolated from Catharanthus roseus grown in highly salt affected coastal region of cuddalore district, Tamilnadu, India four isolates were found to be positive for nitrogenase activity. The isolates were evaluated for their stress tolerance efficiency and screened for different PGP traits. Based on the above studied parameters, and ability to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase (4.24???mol ??-ketobutyrate mg^_1 protein h^_1) the salt tolerant diazotrophic isolate AUM54 was selected for further investigation and identified as Achromobacter xylosoxidans by 16S rRNA gene sequencing. The ability of this isolate to ameliorate salt stress in C. roseus was evaluated under gnotobiotic and pot culture conditions. At 150?mM NaCl level A. xylosoxidans AUM54 treated plants recorded ethylene level of 394.1 p mol ethylene g^?1 FW h^?1 compared to the ethylene level of 516.0 p mol ethylene g^?1 FW h^?1 recorded in the un inoculated control. A. xylosoxidans AUM54 inoculated plants recorded the maximum germination percentage of 98.3, vigor index of 2231.4, plant height of 120.4?cm, root dry weight of 53.24?g Plant^_1 and ajmalicine content of 1.60?mg?g^?1, compared to the germination percentage of 91.6%, vigour index of 1511.5, plant height of 105.8, root weight of 47.2?g Plant^?1, and ajmalicine content of 1.23?mg?g^?1 in uninoculated plants grown without NaCl treatment. This isolate also decreased plant ethylene levels by 11?C23% and increased the antioxidative enzyme content of inoculated C. roseus plants to the tune of 19?C32% for ascorbate peroxidase (APX) activity, 20?C30% for superoxide dismutase (SOD) activity and 4?C16% for catalase (CAT) under normal and salt affected conditions.     

New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata

Isolation of meristematic tissue of the red alga Palmaria palmata by a freezing-thawing method and further maintenance of the tissue in culture showed the existence of groups of meristematic cells in superficial cortical layers of thallus forming wart-like outgrowths. For the first time, proliferations (plantlets) were obtained from meristematic tissue of sporophytic and male gametophytic fronds and tetraspores from submeristematic tissue of sporophytic fronds within a short period (6 weeks). Tissue fragments (1 × 1 mm²) from upper margins of fresh thalli and cell aggregates (10−100,000 cells) from marginal meristem and meristematic warts of fresh thalli and thalli after the freezing-thawing procedure were cultured for getting plantlets. Tissue fragments (TF) and cell aggregates (CA) from submeristematic tissue of fresh thalli were cultured for obtaining tetraspores. For mass getting proliferations (plantlets) and tetraspores we recommend to use CA from marginal tissue of fresh fronds because of fast growth, high numbers of proliferations and simple techniques of the method. The freezing-thawing method allows also to identify meristematic tissue and to obtain plantlets of red algae with apical meristem (e.g., Gelidium spp.).     

Identification and Isolation of Single Cells that Produce Somatic Embryos at a High Frequency in a Carrot Suspension Culture

A system was established in which single cells differentiated to embryos at a high frequency. Small spherical single cells from a carrot (Daucus carota L. cv “Kurodagosun”) cell suspension culture were obtained by fractionation through sieving, using nylon screens and then density gradient centrifugation in Percoll solutions. Eighty-five to 90% of these small single cells differentiated to embryos when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (5 × 10⁻⁸ molar), zeatin (10⁻⁶ molar), and mannitol (0.2 molar) for 7 days, followed by transfer to a medium containing zeatin (10⁻⁷ molar) but no auxin. This indicates that there are at least two phases in the differentiation of embryos from single cells. The progression of the first phase required exogenous auxin, whereas that of the second phase was inhibited by the same growth regulator. The relationship between the morphology and potency for embryogenesis from single cells was discussed. The system established here is a useful one for investigation of differentiation process from a single cell to a whole plant via embryogenesis, especially in its early stage.     

Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10⁴ cells/mL (8.9×10³ cells/cm²) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).     

Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line

The potential of spent medium to support the growth and recombinant protein production of High-Five? cells was investigated. Growth in medium consisting of three parts fresh and one part spent medium was comparable to that in fresh medium (maximal specific growth rates of 0.028 and 0.029 h^?1, and maximal cell densities of 4 and 4.5 × 10⁶ cells ml^?1, respectively). Glucose exhaustion coincided with an abrupt decrease of viability. Of 15 amino acids analyzed, not a single one was completely exhausted at the end of the growth phase. Growth in medium consisting of equal parts spent and fresh medium led to lower maximal cell concentration (2.9 × 10⁶ cells ml^?1) with a smoother death phase. Glucose supplementation at the beginning of the culture or at the end of the growth phase did not lead to an increase of either the maximal cell density or the specific growth rate. Infection of High-Five? cells at three different densities (1.4, 2.5 and 4.2 × 10⁶ cells ml^?1) without medium change led to monotonically decreased specific productions for β-galactosidase. Partial (75%) or total medium replacement at the higher infection density restored the specific production at the levels of the intermediate density infection (321, 292 and 389 U.(10⁶ cells)^?1, respectively).     

Novel biotransformation processes of dihydroartemisinic acid and artemisinic acid to their hydroxylated derivatives by two plant cell culture systems

Novel biotransformation processes of dihydroartemisinic acid (1) and artemisinic acid (2) to their hydroxylated derivatives were investigated using the cell suspension cultures of Catharanthus roseus and Panax quinquefolium crown galls as two biocatalyst systems. Five biotransformation products, 3-α-hydroxydihydroartemisinic acid (3), 3-β-hydroxydihydroartemisinic acid (4), 15-hydroxy-cadin-4-en-12-oic acid (5), 3-α-hydroxyartemisinic acid (6) and 3-β-hydroxyartemisinic acid (7), were isolated by chromatograph methods and identified by the analysis of ¹H NMR, ¹³C NMR, and ESI-MS spectra. Compounds 3–5 were obtained for the first time by biotransformation process. It was also the first time to transform artemisinic acid to yield epimeric 3-hydroxy artemisinic acids in plant cell culture system. The biocatalyst system of C. roseus cell cultures showed a great capacity of regio- and stereo-selective hydroxylation in allyl group of the exogenous substrates. The results also showed that the biocatalyst system of P. quinquefolium crown galls possessed the ability to hydroxylate propenyl group of exogenous substrates in a regio- and substrate-selective manner. Furthermore, the in vitro antitumor activity of the hydroxyl products was evaluated by MTT assay. The result indicated that α-hydroxyl products possessed stronger antitumor activity than β-hydroxyl products against the HepG2 and GLC-82 cell lines.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Pea growth and ion accumulation at varying seed density

Decreased accumulation of elements, particularly of nitrogen, had in dense stand a negative influence on the plants, in spite of the supply of fresh solution and the control of the concentration of nutrients in the pots. The fresh weight and the dry matter of one plant were reduced substantially with rising stand density (from 5 to 10, 20, 40 and 80 plants per 450 sq. cm); the RGR value, the relative absorption rate I_M, the content of all tested elements calculated per one plant, the chlorophyll content in the overground parts of one plant, and the distribution index decrease. Qualitatively the same influence is caused by deficiency of nutrients. Plant dying and self-thinning of the culture occurred in denser stand in the course of cultivation. The root-weight ratio rose with stand densification, particularly at the end of the experiment. The net assimilation rate (NAR) related to chlorophyll dropped with stand density; NAR at density “80” rose slightly from the 22nd day of cultivation and did so at further lower densities. Almost all NAR values lie over the control value at the last measurement. The chlorophyll content in mg g^?1 dry matter of the overground parts rose with stand density to density “40” (41% more than at density “5”); afterwards it dropped. The accumulation (in mg g^?1 d.m.) of phosphorus ions was higher in the plants from dense cultures compared with density “5”, particularly in the roots. The accumulation of potassium was near to the control value (“5”), while that of nitrogen was lower. The utilization quotients of phosphorus and potassium in denser stands were the same or negligibly lower than at density “5”. They were a little higher in nitrogen than in the controls. Also the ratio RGR/I_M was a little higher than in denser stands. The root-weight ratio, the accumulation of elements in mg g^?1 d.m., the chlorophyll content in mg g^?1 d.m., NAR_ch, the utilization quotient, and the ratio RGR/I_M differed qualitatively by the densification of the culture (with complete mineral nutrition) from the influence of element deficiency (at the same stand density).