Studies on the Growth in Culture of Plant Cells: XVIII. NUCLEAR CYTOLOGY OF ACER PSEUDOPLATANUS SUSPENSION CULTURES

Established suspension culture strains of Acer pseudoplatanuswere analysed by chromosome counting and microdensitometricDNA measurements of individual nuclei. Comparison with the root-tipcomplement (2n=4x=52) showed the cultures to be entirely aneuploidwith modal chromosome numbers of approximately 75 and 135 plussome cells with 250–350. Analysis of the frequency distributionsof nuclear DNA values through the growth cycle of a batch cultureshowed that stationary phase cells accumulated in G1 of thecell cycle. Stationary phase DNA distributions could thus beused to indicate the chromosomal status of a culture withoutthe complication of G1 and G2 DNA values for each chromosomenumber. Root-tip and cultured cells showed a close correlationbetween DNA content and chromosome number indicating that structuralchanges and loss of chromosomes had been at random.     

Studies on the Growth in Culture of Plant Cells: XXII. GROWTH LIMITATION BY NITRATE AND GLUCOSE IN CHEMOSTAT CULTURES OF ACER PSEUDOPLATANUS L.

The responses of steady-state cell populations of Acer pseudoplatanusL. (sycamore) in chemo-stats to changes in the nitrate concentrationof their environment was generally predictable by equationsderived for ideal, free-cell, nitrate-limited populations. Astep-down in nitrate concentration in the input medium of achemostat produced interlocking oscillations in spent-mediumnitrate, intracellular pools of nitrate and amino-N, proteinaccumulation, and cell division. The rate of nitrate uptakeinto the cell and thus the flow of nitrogen into protein (andultimately the specific growth rate) was seen to be finely regulatedby spent-medium nitrate concentration. Steady states of growth of sycamore cells were established withglucose as the limiting nutrient both from inoculation and afterswitching from nitrate to glucose limitation. Glucose-limitedcells produced by glucose step-down of a nitrate-limited populationsuffered major losses of cell material, including starch andcell wall polysaccharide, to the extent that protein then accountedfor c. 70% of cell dry weight.     

Studies on the Growth in Culture of Plant Cells: XIV. CARBOHYDRATE OXIDATION DURING THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

Marked changes in the activities of the Embden–Meyerhof–Parnas(EMP) pathway and the pentose phosphate pathway occur duringthe progress of growth of sycamore cells in batch suspensionculture. The activities of the two pathways were investigatedby measuring the activities of six EMP pathway and four pentosephosphate pathway enzymes, and by determining the contributionsof ¹⁴C from (1-¹⁴C)- and (6-¹⁴C)-glucose to CO₂. During theearly stages of culture both the EMP pathway and the pentosephosphate pathway make appreciable contributions to carbohydrateoxidation, but following the initiation of cell division, carbohydrateoxidation is predominantly via the EMP pathway. All the enzymesassayed were found to be associated with the soluble fractionof the cells. The regulation of carbohydrate oxidation in sycamorecells and the possible role of the pentose phosphate pathwayin supplying NADPH for biosynthesis are discussed.     

Studies on the Growth in Culture of Plant Cells: XIX. CHANGES IN THE LEVELS OF FREE AND MEMBRANE-BOUND POLYSOMES DURING THE GROWTH OF ACER PSEUDO PLA TAN US CELLS IN BATCH SUSPENSION CULTURE

Techniques have been established which give reproducible yieldsof total and of free and bound polysoines from cultured sycamorecells liarvested at intervals throughout the cycle of growthfollowed in batch culture. High levels of polysoines build upin the cells during lag phase and persist into early exponentialgrowth. Later in the growth cycle, although the ribosomal materialper unit volume of culture continues to rise, the content percell of ribosomes and polysomes progressively declines untilthe cells enter the stationary phase. There is also a characteristicpattern of change in the relative proportions of free and boundpolysomes throughout the growth cycle of the cultures. Bothfractions contain different but significant levels of ribosomalsubunits and monomers. The RNAs released from the free polysoineshad a greater amount of poly(A) sequences than that from thebound polysomes. These findings are discussed in relation tothe changing metabolic activities of the cells which occur duringtheir progress through batch culture.     

Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

Studies on the Growth in Culture of Plant Cells: XI. THE INFLUENCE OF SHAKING RATE ON THE GROWTH OF SUSPENSION CULTUEES

The influence of shaking rates (expressed as revolutions permin) on orbital shaking platforms (1 in (2.54 cm) diam. rotarymotion) on the growth of cell suspension cultures of Acer pseudoplatanusL. and Atropa belladonna cultivar lutea Döll are described.By following cell growth and respiration and the levels of oxygenand carbon dioxide in the media during the progress of incubationit is concluded that the reduction of growth at sub-optimalshaking rates is not due to oxygen deficiency or toxic accumulationof carbon dioxide. The growth of the Atropa cell suspensionin ‘closed systems’ has been studied by the developmentof modified culture vessels and evidence obtained that the reducedgrowth in the systems is due to the formation by the culturesof an unidentified volatile growth inhibitor and not to eitheroxygen depletion or toxic accumulation of either carbon dioxideor ethylene. It is suggested that the reduced growth in ‘opensystems’ cultures at sub-optimal shaking speeds is eitherdue to retention of this volatile inhibitor or to restrictionof nutrient uptake by the existence of a stationary liquid-phaseboundary to the cells.     

Studies on the Growth in Culture of Plant Cells: III. CHANGES IN FINE STRUCTURE DURING THE GROWTH OF ACER PSEUDOPLATANUS, L. CELLS IN SUSPENSION CULTURE

A technique has been developed for the electron microscope studyof the free cells and small cell aggregates of suspension culturesof Acer pseudoplatanus, L. Changes in fine structure have beenfollowed during the growth of a batch culture over 24 days,covering the lag phase, the phase of exponential growth, andthe stationary phase to a condition where the cells show evidenceof senescence. During the lag phase there is a massive synthesisof new cytoplasm and an increase in the number of mitochondriaand ribosomes. By the point of transition to the phase of exponentialgrowth many of the ribosomes are either attached to the ER membranesor are organized in spherical or spiral clusters. Multivesicularbodies are frequently observed. The development of the cellplate can be followed in some detail at this stage. As the rateof cell division decreases and cell enlargement begins the cytoplasmcomes to constitute a thin lining layer with fewer ribosomes,less prominent ER membranes and apparently fewer mitochondria.At this time starch begins to form and the frequency of lipid(or protein) bodies and of membrane enclosed crystals increases.During the stationary phase, which begins at about the 15thday of culture, the old cell walls show characteristic changesand are frequently ruptured. Intra-cytoplasmic vacuoles appearand then with the continuation of culture disappear as the cytoplasmiclayer approaches its minimum thickness. Nuclei show invaginationsand these often contain characteristic ‘aged’ mitochondria.     

Studies on the Growth in Culture of Plant Cells: XXIV. EFFECTS OF 2, 4-DICHLOROPHENOXYACETIC ACID AND LIGHT ON THE GROWTH AND METABOLISM OF ACER PSEUDOPLATANUS L. SUSPENSION CULTURES

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore (AcerpseudoplatanusL.) caused growth cessationafter an initial period of exponential growth. The presenceof white light reduced the amount of growth after 2,4-D withdrawal.Growth cessation, in the absence of 2,4-D, was accompanied bythe accumulation of the free amino acid, serine. This accumulationbegan before the cessation of growth and was rapidly reversedby the re-addition of 2,4-D.     

Studies on the Growth in Culture of Plant Cells: XV. UPTAKE AND UTILIZATION OF URIDINE DURING THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

[2-¹⁴C]-uridine is rapidly taken up by sycamore cells in suspensionculture. A proportion of the radioactivity enters RNA withoutmeasurable delay, whilst the remainder equilibrates with a largepool of phosphorylated compounds, the major radioactive componentof which is 5'-UMP. Both the uracil and cytosine residues ofRNA receive label from [¹⁴C]-uridine and, when the cells aresupplied with high concentrations of uridine, these bases arederived almost exclusively from the nucleoside. [¹⁴C]-uridine is incorporated into RNA at all stages of thegrowth cycle of batch cultures; its continuing incorporation,when the total RNA content of the cells is rapidly decreasing,indicates a high rate of turnover of the total RNA. Long-termlabelling experiments also indicate turnover of RNA during thephase of active cell division and suggest that a large proportionof the degradation products are not re-utilized for RNA synthesis. Sycamore cells degrade [2-¹⁴C]-uridine with release of ¹⁴CO₂.The proportion degraded increases from 25 per cent at an externaluridine concentration of 10^–6M to 75 per cent at 10^–3M. Despite this, nucleic acids are the only macromolecules thatreceive a significant amount of radioactivity from [2-¹⁴]C-uridine.     

Studies on the Growth in Culture of Plant Cells: II. CHANGES IN RESPIRATION RATE AND NITROGEN CONTENT ASSOCIATED WITH THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

Changes in nitrogen content and in respiration rate have beeninvestigated in cell suspension cultures of Acer pseudoplatanus.Nitrogen content and rate of oxygen uptake rise sharply earlyin the period of culture, during which there is no significantincrease in dry weight and only a small increase in cell number.During the subsequent period of rapid cell division there isa decline in both respiration rate and nitrogen content permg dry weight or per cell. Pronounced rises in respiration rateand cell nitrogen therefore occur prior to the period of rapidcell division. The strong correlation between nitrogen contentand oxygen consumption suggests that the respiration rate ismuch more closely related to changes in protein content thanto changes in cell number, dry weight, or packed-cell volume.     

Studies on the Growth in Culture of Plant Cells: IX. ADDITIONAL FEATURES OF THE FINE STRUCTURE OF ACER PSEUDOPLATANUS L. CELLS OULTURED IN SUSPENSION

Extensive ridge-like structures lining the outer walls of cellsat the surface of cell aggregates and less elaborate thickeningson the walls between adjacent cells are reported and comparedwith the wall thickenings observed in ‘transfer cells’.Mitochondria with an atypical crista are described. Plastidsin cells cultured in the standard synthetic medium contain poorlydeveloped internal lamellae and function as amyloplasts duringthe growth cycle. Their differentiation into chloroplasts isinduced by replacing the 2,4-D by NAA in the medium. A structureis proposed for the crystalloid core of the microbodies, whichare a prominent feature of stationary phase cells.     

Studies on the Growth in Culture of Plant Cells: XVII. ANALYSIS OF THE CELL CYCLE OF ASYNCHRONOUSLY DIVIDING ACER PSEUDOPLATANVS L. CELLS IN SUSPENSION CULTURE

Three methods of cell cycle analysis, involving the use of tritiatedthymidine, have been applied to asynchronously dividing suspensioncultures of sycamore. Conditions for an effective chase of unlabelledthymidine were established from a study of the kinetics of entryand incorporation of tritiated thymidine into the cells. Thelevels of thymidine used did not affect the rate of cell divisionor the duration of the phases of the cell cycle. The analyses of the cell cycle based upon pulse labelling, continuouslabelling, and a combination of densitometry and autoradiographywere in good agreement and showed that the phases S (mean 7.0h), G2 (mean 8.5 h) and mitosis (mean 3.0 h), were of relativelyconstant duration, whereas G1 was of variable duration. No relation between nuclear DNA content and mitotic-cycle timeor the duration of S-phase could be inferred from the data presented.     

Studies of the Growth in Culture of Plant Cells

Mitochondria have been isolated from sycamore cells (Acer pseudoplatanusL.) grown in suspension culture, and resemble those of otherplant tissues. Malate, succinate, and NADH are oxidized withrespiratory control. The respiration is partially inhibitedby antimycin A and KCN, but not by amytal and rotenone. Octylguanidine,oligomycin, and uncouplers all affect the coupled respiration. The proportion of the respiration resistant to KCN was foundto change during the life of the culture, being greatest duringthe lag phase and least during the linear phase. The relationshipof these changes in the electron transport pathways to the changingdemand of the culture for phosphorylated and other intermediatesis discussed.     

Studies on the Growth in Culture of Plant Cells: XVI. NITROGEN ASSIMILATION DURING NITROGEN-LIMITED GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN CHEMOSTAT CULTURE

Cultures of Acer pseudoplatantis L. cells have been establishedin a chemostat with nitrogen as the limiting nutrient factor.During prolonged growth at three different dilutions severalenzymes concerned with the assimilation of nitrate and ureafrom the culture medium achieved steady states of activity.Neither the enzyme activities nor the amino-acid contents ofthe cells showed marked changes with change in dilution underthe nitrogen-limiting conditions employed here. One of the steady states was perturbed by supplementing theculture medium with gluta-mate as an additional nitrogen source.This caused a rapid and considerable enhancement of the alaninecontent of the cells, presumably resulting from transaminationbetween pyruvate and the incoming glutamate. In the longer term,the transition caused an elevation of the levels of enzymesconcerned with the diversification of nitrogen from glutamate(glutamate-oxaloacetate and glutamate-pyruvato transaminasesand -glutamyl transferase), whilst enzymes concerned with glutamateformation (urease and, most probably, nitrate reductase) declinedin activity     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

Studies on the Growth in Culture of Plant Cells: XII. A VERSATILE SYSTEM FOR THE LARGE SCALE BATCH OR CONTINUOUS CULTURE OF PLANT CELL SUSPENSIONS

A versatile large-scale batch culture unit has been developedfor the growth of plant cell suspension cultures. This unithas been modified to permit of intermittent or continuous renewalof culture medium and, in a modified form, incorporated intoopen continuous culture systems of the chemostat and turbidostattype. A fully automatic culture sampler has been incorporatedinto the basic culture unit. The culture systems described havebeen successfully operated using a cell suspension derived fromAcer pseudoplatanus and results are presented demonstratingsynchronous growth in batch culture, prolonged logarithmic increassein cell number under conditions of high aeration and culturemedium renewal, and steady states of growth resulting from automaticregulation of the optical density of the cell suspension andfrom fixed rates of displacement of cell suspension by new medium.The potentialities of the culture systems are discussed in thelight of the experimental results presented.     

Studies on the Growth in Culture of Plant Cells: VII. EFFECTS OF KINETIN ON THE CARBOHYDRATE AND NITROGEN METABOLISM OF ACER PSEUDOPLATANUS, L CELLS GROWN IN SUSPENSION CULTURE

Aspects of the carbohydrate and nitrogen metabolism of Acerpseudcplatanus, L cell suspension cultures grown on a syntheticmedium containing 2 per cent glucose and 1.0 mg/l 2,4-dichlorophenoxyaceticacid and kinetin either at 0.25 mg/l (low kinetin) or at 2.5mg/l (high kinetin) are described. High kinetin inhibits growthas measured by increase in cell number, packed-cell volume,and cell dry weight. Although not inhibitory to glucose utdization,high kinetin inhibits the O₂ uptake of the cells. Such cellscontain only a trace amount of fructose and their rate of O₂uptake can be raised to that of the low kinetin cells by a periodof fructose feeding. The O₂ uptake of both kinds of cell issensitive to malonate but the stimulation of O₂ uptake inducedby bis(hexafiuoroacetonyl)-acetone (‘1799’) at 0.2mM is much less with the high-kinetin than the low-kinetin cells.The enzymes phosphoglucoseiseomerase and glucose-6-phosphatedehydrogenase are much less active in the high-kinetin cells.Mitochondria isolated from both kinds of cells show good respiratorycontrol although slightly lower values for Q_O2(N), ADP/O ratioand control ratio are recorded with mitochondria from the highkinetin cells. Kinetin at 2.5 mg/l slightly reduces the ADP/Oratio of isolated mitochondria but at 4.0 mg/l their responseto ADP is completely suppressed. Extracellular hemicelluloseformed in presence of high kinetin has a reduced content ofgalactose and xylose and an increased content of glucose. Theseobservations indicate that the inhibition of respiration byhigh kinetin is mainly due to suppression of glucose conversionto other sugars rather than to inhibition of glycolysis or terminalrespiration. High kinetin decreases the rate of protein but not of amino-acidsynthesis. Suppression of the synthesis of particular proteinsmay be an important factor responsible for the reduced cellyield of the cultures in presence of high kinetin. The significanceof these observations to our understanding of the critical metaboliceffects of cytokinina is discussed. Acer pseudoplatanus cells release amino acids into their culturemedium early in the period of batch culture and largely reabsorbthem as incubation proceeds.     

PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS : I. Establishment of Long-Term Growth in Culture and Studies of Differentiated Properties

Rat sympathetic ganglia were disrupted by mechanical agitation to yield dissociated primary neurons, and the conditions for long-term growth in culture of the isolated neurons were examined. The neurons were grown with or without non-neural cells, simply by the addition or deletion of bicarbonate during growth in culture. Fluorescence histochemistry indicated that the isolated neurons contained catecholamines; incubations with radioactive precursors were used to verify the synthesis and accumulation of both dopamine and norepinephrine. The neurons also produced octopamine using tyramine as precursor, but not with tyrosine as the precursor. In the presence of eserine, older cultures synthesized and stored small amounts of acetylcholine. The cultures did not synthesize and accumulate detectable levels of radioactive γ-aminobutyric acid, 5-hydroxytryptamine, or histamine.     

Studies on the Growth in Culture of Plant Cells: V. LARGE-SCALE CULTURE OF ACER PSEUDOPLATANUS L. CELL SUSPENSIONS

A technique is described for the large-scale culture of sycamorecell suspensions. Two 10-1 wide-mouthed flat-bottomed culturebottles are spun continuously at 120 rpm inclined at 45°from the vertical. Each bottle contains a 4.5 1 culture which,after 21 d at 25 °C yields a suspension of 10⁹ cells, 1.41 of packed-cell volume and 40 g dry weight. The growth characteristicsof the cultures are described and it is demonstrated that largevolumes of suspension can be withdrawn during incubation withoutaltering the general growth pattern or the yield of cells perunit volume of the final culture.     

Studies on the Culture Conditions of Higher Plant Cells in Suspension Culture

To study the influence of cultural conditions on higher plant cells in suspension culture, the effects of nutritional conditions on the growth of suspended cells were investigated. Calluses were induced from 39 species of Nicotiana plants and 6 species of Populus plants on agar slant media, then these were transferred to suspension cultures. Concentrations of 2,4-D and kinetin suitable for incubation of callus from each plant were investigated and species having high growth rates in the appropriate medium were selected.

The effects of concentrations of auxins and kinetin, a variety of carbon and nitrogen sources, thiamin and myo-inositol on growth of the selected calluses were also studied. Of these calluses studied, N. glutinosa, N. tabacum var. Xanthi ova and P. hybrids were selected as calluses having high growth rates. Myo-inositol had no effect on any callus growth, and thiamin gave a distinct effect on Populus callus only. Nitrate as a nitrogen and sucrose as a carbon sources, and 2,4-D as an auxin were most effective in all calluses studied. Kinetin was essential for N. glutinosa among the calluses studied. Although high sugar concentrations tended to lengthen the lag period in the growth curve, there was no difference in the growth rates of the logarithmic phase among the concentrations.