Growth Of Cell Suspension Cultures Research Articles

Population responses in tobacco cell cultures during selection for resistance to para-fluorophenylalanine

Growth of cell suspension cultures of Nicotiana tabacum L. line TX1 is strongly inhibited by concentrations of para-fluorophenylalanine (PFP) >300 μM. Through step-wise selection, lines capable of growing in 450 μM PFP can readily be obtained. The manner in which the initial resistant variant population arises, however, is not consistent with a model involving rare appearance of individual mutant cells within the population. The latter model is also not supported by the results of reconstruction experiments which show that a very substantial subpopulation of resistant cells must be present in order to allow a growth response in the presence of the selection agent.

Effect of Phaseolotoxin on the Synthesis of Arginine and Protein

Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [(14)C] ornithine into protein-arginine of the mesophyll cells. By contrast more than 80% of the [(14)C]ornithine supplied to untreated tissue was incorporated into protein-arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [(14)C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O(2) evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue.

Somatic embryogenesis in cell cultures of Glycine species

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

Feruloylated pectins from the primary cell wall: their structure and possible functions

Primary cell walls from exponentially growing cell-suspension cultures of spinach contained ferulic acid and p-coumaric acid esterified with galactopyranose and arabinopyranose residues of polysaccharides. The feruloylated polysaccharides behaved in exactly the same way as total cell-wall pectin with respect to (1) extraction with chelating agents, (2) extraction by trans-elimination degradation, (3) extraction with mild acid, and (4) electrophoretic separation into acidic and neutral species. Partial digestion of cell walls with Driselase, under conditions which specifically inhibited galactanase and galactosidases yielded galactose-containing feruloyl tri- to pentasaccharides, in all of which the feruloyl group was on the non-reducing terminus. Larger feruloyl oligosaccharides were also found, some of which were acidic. Partial acid-hydrolysis of cell walls gave a homologous series of feruloyl oligosaccharides, probably with the structure Feruloyl-arabinopyranose-(arabinofuranose)n-arabinose where n=0-7. Evidence is presented that the arabinose chain was unbranched, with the feruloyl group on the nonreducing terminus. It is suggested that acidic and neutral pectins carry ferulic acid on the non-reducing termini of the neutral arabinose- and/or galactose-containing domains. The pectins carry approximately one feruloyl residue per 60 sugar residues. Possible rôles of feruloyl pectin in the regulation of cell expansion, in disease resistance, and in the initiation of lignification are discussed.

Preparation of viable protoplasts from suspension-cultured loblolly pine (Pinus taeda) cells and subsequent regeneration to callus

Protoplasts were prepared from exponentially growing cell suspension cultures derived from Pinus taeda seedlings. The cell suspension was derived from callus that had been initiated on solid Murashige and Skoog medium supplemented with 2mg/1 2,4-D, and maintained in liquid LM medium (D.C. Verma et al, Proceedings of the 5th International Congress of Plant Tissue and Cell Culture, Tokyo, Japan, 1982, p. 59–60). Evaluation of a range of enzyme formulations showed that almost all cells could be converted to viable protoplasts in 9 hours with the combination of 0.5% each of Onozuka R-10, Macerozyme R-10, Hemicellulase, Driselase, and bovine serum albumin in osmoticum (0.4 M mannitol, 0.2 M sorbitol, 1.5 mM CaCl2, 0.7 mM KCl). Protoplast purification required only simple washing which was achieved without damaging centrifugation. When cultured in LM medium supplemented with 1.5mM CaCl2, 10 mM arginine, 10mM glutamine and 5 mM asparagine, cell wall formation was observed after 48 hours. The cultured protoplasts produced numerous colonies in 3 weeks which continued to grow in liquid medium.

Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharide.

1. Cell walls from rapidly growing cell suspension cultures of Spinacia oleracea L. contained ferulic acid and p-coumaric acid esterified with a water-insoluble polymer. 2. Prolonged treatment with trypsin did not release may feruloyl esters from dearabinofuranosylated cell walls, and the polymer was also insoluble in phenol/acetic acid/water (2:1:1, w/v/v). 3. Treatment of the cell walls with the fungal hydrolase preparation "Driselase' did liberate low-Mr feruloyl esters. The major esters were 4-O-(6-O-feruloyl-beta-D-galactopyranosyl)-D-galactose and 3?-O-feruloyl-alpha-L-arabinopyranosyl)-L-arabinose. These two esters accounted for about 60% of the cell-wall ferulate. 4. It is concluded that the feruloylation of cell-wall polymers is not a random process, but occurs at very specific sites, probably on the arabinogalactan component of pectin. 5. The possible role of such phenolic substituents in cell-wall architecture and growth is discussed.

Selective guanosine phosphate deficiency in hepatoma cells induced by inhibitors of IMP dehydrogenase.

Inhibition of IMP dehydrogenase in AS-30D hepatoma cells in suspension culture resulted in a pronounced and selective reduction of guanine nucleotide pools. Total acid-soluble guanine nucleotides decreased to 40% and the content of GTP and GDP dropped to about 20% of control within 4 h when mycophenolate or ribavirin were used as the inhibitors. Induction of GTP deficiency was associated with a 50% rise in UTP and other uracil nucleotides. Guanosine rapidly reversed both the reduction of guanine nucleotide pools and the elevation of cellular UTP contents. Enzymatic nucleotide analyses in cell and tissue extracts after treatment with ribavirin indicated that ribavirin 5'-triphosphate was an effective substrate for yeast hexokinase, yeast phosphoglycerate kinase, and nucleosidediphosphate kinase from yeast or bovine liver. These results were confirmed in detail by the use of synthetic ribavirin 5'-triphosphate and 5'-diphosphate. The latter nucleotide analog was also a substrate of pyruvate kinase from muscle. Mycophenolate-induced GTP deficiency was associated with an arrest of hepatoma cell growth in suspension culture. Ribavirin, at an equimolar concentration, was much less effective in this respect. None of the two inhibitors had a detectable effect, however, in vivo when guanine or uracil nucleotides were assayed in liver. This indicated that an inhibition of de novo guanylate synthesis in vivo can be compensated by salvage pathway synthesis.

Psoralen plus ultraviolet radiation-induced inhibition of DNA synthesis and viability in human lymphoid cells in vitro.

Abstract— 8‐Methoxypsoralen (8‐MOP) plus high intensity long wavelength ultraviolet radiation (UV‐A) is presently being used to induce remissions of psoriasis and mycosis fungoides. Previous studies demonstrated inhibition of DNA synthesis in circulating leukocytes from some patients during this therapy. The present study is designed to determine whether conditions of 8‐MOP concentration and UV‐A exposure attained during therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (3HTdR) incorporation and cell growth in suspension culture following UV‐A exposure alone or with therapeutic concentrations of 8‐MOP was examined in peripheral blood lymphocytes and in Ebstein‐Barr virus transformed human lymphoblas‐toid cell lines. UV‐A exposure alone induced a dose‐dependent inhibition of HTdR incorporation in both types of lymphoid cells (3000 J/m2 resulted in 77% of control 3HTdR incorporation). Pre‐incubation with 0.1 μg/m/ 8‐MOP before UV‐A exposure induced a significantly greater inhibition of 3HTdR incorporation (3000 J/m2 resulted in 61% of control 3HTdR incorporation). Greater inhibition of 3HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0μg/mC 8‐MOP (3000 J/m2 resulted in 41% of control) but not in the lymphocytes (3000 J/m2 resulted in 63% of control). The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV‐A alone (29,000 J/m2) but preincubation with 0.1 μg/mC 8‐MOP resulted in 40% survival after 3000 J/m2 (D37 approximately 3000 J/m2) and preincubation with 1.0 μg/ 8‐MOP resulted in 0.6% survival after 3000 J/,2 (D37 approximately 800 J/m2). This study suggests that the low doses of 8‐MOP and UV‐A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. The long term consequences of such damage remain to be determined.

The influence of applied carbohydrates on the growth and greening of cultured spinach ( Spinacia oleracea L.) cells

When the sucrose concentration in the culture medium used for the growth of cell suspension cultures of spinach was at 2.5 mg/ml or higher, chlorophyll synthesis was inhibited. Other readily utilisable sugars (glucose, fructose, maltose) in contrast to slowly utilised sugars (inulin, raffinose) similarly inhibited chlorophyll synthesis. Analysis of the spent medium suggests the absence of appreciable free-space invertase activity. Measurement of the oxygen exchange rates of the cells in light and in darkness indicated that applied sucrose, in contrast to its monosaccharide components, directly inhibited photosynthesis or promoted photorespiration.

Divergent Patterns of Marrow Cell Suspension Culture Growth in the Myeloid Leukemias: Correlation of In Vitro Findings With Clinical Features

Cellular recovery, maturation, and colony-forming cell (CFC) generation patterns of bone marrow cells from 23 patients with acute, subacute, and chronic myeloid leukemia (AML, SML, and CML) were studied using liquid and agar culture techniques. Increased recovery of proliferative myeloid cells from liquid culture was noted in 6 of 8 AML patients at diagnosis or relapse and 5 of 7 untreated SML patients. Patients with either AML or SML with rapid clinical progression exhibited greater recovery of cells in vitro with less maturation than patients with more stable disease. Studies from 3 patients with CML showed normal to increased cellular recovery with normal maturation. Three of 4 studies of AML patients followed sequentially in apparent remission, but with impending relapse, exhibited increased numbers of myeloblasts and promyelocytes, whereas 28 of 32 studies performed during stable remission were normal. The normally observed increase in CFC during liquid culture was absent in most leukemic marrow samples studied (3 of 4 AML, 4 of 6 SML, and 2 of 3 CML). Persistent low recovery of CFC during AML remission was associated in 3 patients with short remission duration. These studies indicated the potential utility of these techniques for the clinical evaluation of patients with myeloid leukemia and for studying factors involved in the progression of these diseases.

Divergent patterns of marrow cell suspension culture growth in the myeloid leukemias: correlation of in vitro findings with clinical features

Cellular recovery, maturation, and colony-forming cell (CFC) generation patterns of bone marrow cells from 23 patients with acute, subacute, and chronic myeloid leukemia (AML, SML, and CML) were studied using liquid and agar culture techniques. Increased recovery of proliferative myeloid cells from liquid culture was noted in 6 of 8 AML patients at diagnosis or relapse and 5 of 7 untreated SML patients. Patients with either AML or SML with rapid clinical progression exhibited greater recovery of cells in vitro with less maturation than patients with more stable disease. Studies from 3 patients with CML showed normal to increased cellular recovery with normal maturation. Three of 4 studies of AML patients followed sequentially in apparent remission, but with impending relapse, exhibited increased numbers of myeloblasts and promyelocytes, whereas 28 of 32 studies performed during stable remission were normal. The normally observed increase in CFC during liquid culture was absent in most leukemic marrow samples studied (3 of 4 AML, 4 of 6 SML, and 2 of 3 CML). Persistent low recovery of CFC during AML remission was associated in 3 patients with short remission duration. These studies indicated the potential utility of these techniques for the clinical evaluation of patients with myeloid leukemia and for studying factors involved in the progression of these diseases.

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

AbstractA method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two‐tier culture vessel the growth medium with the cells was separated from the CO2 reservoir. Definite CO2 concentrations were established by a K2CO3/KHCO3 buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO2 level. At 0.5% CO2 the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO2 an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight.The photoautotrophic growth was also greatly influenced by the 2,4‐D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10−8M 2,4‐D.The photosynthetic activity of the cells was measured by the light dependent 14CO2 incorporation. At 0.5% CO2 the cell suspensions assimilated about 100 μmol CO2/mg chlorophyll × h. In the presence of 1% CO2 the light driven assimilation was raised up to 185 μmol CO2/mg chlorophyll × h. In both cases, the dark incorporation of CO2 was merely 1.8% of the values obtained in light.

Aspartate carbamoyltransferase inhibition and uridylate trapping result in a synergistic depression of uridine triphosphate in hepatoma cells

The induction of lethal UTP deficiency by uridylatetrapping sugar analogs [ 1 ] depends on a relatively low rate of pyrimidine nucleotide biosynthesis [2]. Increased activities of enzymes involved in the de novo synthesis of pyrimidine nucleotides have been demonstrated in a spectrum of hepatomas [3,4]. The resistance of solid hepatomas [5] and of ascites hepatoma cells to the action of D-galactosamine (GalN) has been explained by the efficient compensation of uridylate trapping by de novo synthesis resulting in only a limited depression of UTP [6]. Noncompensated uridylate trapping can be induced by the synergistic action of GalN and an inhibitor of de novo pyrimidine synthesis. A potent inhibitor of aspartate carbamoyltransferase (EC 2.1.3.2) has been introduced recently [7-lo]. N-(Phosphonacetyl)-Laspartate (PALA) has been synthesized by Collins and Stark [7] as a transition state intermediate analog of the aspartate carbamoyltransferase reaction and inhibits the enzyme in a number of cells and tissues [8,9]. PALA is a competitive inhibitor with respect to carbamoylphosphate and has a Ki of about 10W9 mol/l for the enzyme from mammalian cells [9,10]. This paper demonstrates the depression of de novo uridylate synthesis and UTP contents by PALA in ascites hepatoma cells. The depression of UTP by either PALA or GalN is insufficient, however, for a complete and irreversible inhibition of hepatoma cell growth in suspension culture. Only the combination of GalNinduced uridylate trapping with the blockage of de novo pyrimidine synthesis results in severe UTP deficiency and cellular death.

Reversible loss in suspension culture of a major cell-surface glycoprotein of the TA3-Ha mouse tumor.

A major cell-surface glycoprotein of the TA3-Ha ascites mammary adenocarcinoma diminished during transfer from ascites growth to cell growth in suspension culture. A sensitive, hemagglutination-inhibition assay that used a lectin from Vicia graminea seeds indicated approximately a 50% loss after 7-10 days of culture and a 90% loss after 2 months. These findings were corroborated by carbohydrate and amino acid analysis with gas-liquid chromatography of trypsin glycopeptides released from the cell surface. Repassage of the cultured cells in vivo caused the reappearance of the surface glycoprotein.

Effects of Chemical and Physical Conditions on Growth ofCamptotheca acuminataCell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA3, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.

Effects of Chemical and Physical Conditions on Growth of Camptotheca acuminata Cell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA3, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.

Effects of environmental changes on sugars, tannins, and organized growth in cell suspension cultures of white spruce

Callus from hypocotyls of white spruce (Picea glauca [Moench] Voss) was grown on agar under defined conditions with high levels of calcium nitrate. Transfer of callus to liquid suspension cultures and maintenance of suspensions either under a regime of constant temperature and light or under alternating conditions similar to those of a late spring day, affected the content of free sugars, tannins, and aldehydes. Under the alternating conditions the levels of these substances increased greatly compared to those under the constant environment. By contrast, vascularization of cell clumps, which was comparable to the differentiation of hypocotyls in seedlings, was obtained only under constant conditions. Cells at the centre of the clumps developed secondary wall thickenings and bordered pits, and were surrounded by cambial-like initials.

Studies on the Growth in Culture of Plant Cells

The influence of shaking rates (expressed as revolutions per min) on orbital shaking platforms (1 in (2.54 cm) diam. rotary motion) on the growth of cell suspension cultures of Acer pseudoplatanus L. and Atropa belladonna cultivar lutea Döll are described. By following cell growth and respiration and the levels of oxygen and carbon dioxide in the media during the progress of incubation it is concluded that the reduction of growth at sub-optimal shaking rates is not due to oxygen deficiency or toxic accumulation of carbon dioxide. The growth of the Atropa cell suspension in ‘closed systems’ has been studied by the development of modified culture vessels and evidence obtained that the reduced growth in the systems is due to the formation by the cultures of an unidentified volatile growth inhibitor and not to either oxygen depletion or toxic accumulation of either carbon dioxide or ethylene. It is suggested that the reduced growth in ‘open systems’ cultures at sub-optimal shaking speeds is either due to retention of this volatile inhibitor or to restriction of nutrient uptake by the existence of a stationary liquid-phase boundary to the cells.

Callus and cell suspension culture of bush bean (Phaseolus vulgaris)

A solid medium was developed for callus cultures originating from explants of root, hypocotyl, and cotyledon of seedlings of bush bean, and a liquid medium was developed for the growth of cell suspension cultures derived from the callus cultures. Some unsatisfactory media are recorded. Concentrations of mineral salts for cell suspension cultures are lower than for callus cultures. Both coconut milk and other organic substances are required for maximum growth. With cell suspensions the effect of deproteinized coconut milk is the same as that of raw coconut milk but, with callus cultures, deproteinized coconut milk gives greater yield. There are no obvious differences in yield of callus derived from root, hypocotyl, or cotyledon. Few differences in yield were obtained between cell suspension cultures from root, hypocotyl, and cotyledon but those from root gave the highest yield in dry weight. However, in the same medium, cells from the three origins are very similar in form and appearance. Some effects of different media on cell form and clumping are described. The yield in suspension culture is very high. Increase in cell number, fresh weight, and dry weight is about 100-fold in 12 days involving about six to seven divisions per cell.