Studies on the Growth in Culture of Plant Cells: I. GROWTH PATTERNS IN BATCH PROPAGATED SUSPENSION CULTURES

Techniques are described for following increases in total cellnumber, fresh weight and dry weight, and changes in mean cellsize, and in the relative number of free cells to cell aggregatesduring the growth of batch-propagated suspension cultures oftissues derived from several species of angiosperms. When totalcell number is plotted against time it is seen that there canbe distinguished in sequence a lag phase, phases of acceleration,maximum rate, and negative acceleration of cell division and,finaly, a stationary phase. Studies with Parthenocissus tricuspidatacrown-gall tissue, growing in a synthetic liquid medium, haveshown that the total cell production per culture in the firstinstance is limited by nitrate supply rather than by the supplyof other inorganic ions, sucrose supply aeration, or the releaseof endogenous inhibibors. Studies, particularly with Acer pseudoplatanustissue, have shown that during the period of high cell-divisionrate, mean cell size reached its minimum value and average numberof cells per cell aggregate its maximum value. Cell separationdoes not occur to a significant extent until cell-division activityhas almost ceased and it is dependent upon cell expansion. Thebalance between cell division and cell expansion determinesthe ‘cellular unit’ composition of the cultures.Refinement of the control of growth patterns in plant suspensioncultures calls for further study of the ‘conditioning’of media, of factors which limit the duration of the periodof high mitotic activity, and of the conditions necessary forfull and rapid cell expansion.     

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: 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: 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: 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: 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: 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.     

Growth Inhibition Studies: I. MEASUREMENT OF THE EFFECTS OF INHIBITORS ON THE GROWTH RATE OF FLAX SEEDLINGS

A method for measuring the rate of growth of small seedlingsunder special conditions suitable for inhibition and reversalstudies is described, and its use is illustrated in studiesof the inhibitory action of a variety of chemical substances. The limitations imposed, in such studies, by the osmotic pressureof the test solutions have been examined in the case of flaxseedlings.     

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: 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: 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 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.     

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: X. FURTHER STUDIES ON THE CONDITIONING OF CULTURE MEDIA BY SUSPENSIONS OF ACER PSEUDOPLATANUS L. CELLS

The standard synthetic culture medium (Stuart and Street, 1969)has been modified by adjustment of its initial pH to 6.4 andby the addition of gibberellic acid (0.25 mg/l) and of a mixtureof 15 L-amino acids formulated from an analysis of the conditionedmedium. The minimum effective density for the growth of sycamorecell suspensions in the standard medium is 9–15 x 10³cells ml^–1, for the modified synthetic medium it is 2.0x 10³ cells ml^–1, and for conditioned medium 1.0–1.25x 10³ cells ml^–1. Using either conditioned medium (Stuart and Street, 1969) orthe modified synthetic medium it is demonstrated that the growthof cultures initiated at low density is enhanced by a volatilefactor released from actively growing cell suspensions. In presenceof conditioned medium and this volatile factor cultures canbe established from stationary-phase cells at a density of 6x 10² cells ml^–1. The volatile factor can be absorbedin 40 per cent w/v KOH but attempts to replace the factor byair containing carbon dioxide at concentrations up to 5 percent have so far been unsuccessful.     

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: 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     

The Role of Boron in Plant Growth: II. THE EFFECT ON GROWTH OF THE RADICLE

Estimates of cell number, cell volume, respiratory rate, nitrogen,sugar, and nucleic acid content were made on 1 mm. sectionsof the radicles of field bean at frequent intervals during thefirst 96 hours of growth in nutrient solutions with and withoutboron. The primary effects of deficiency were cessation of cell divisionand enlargement of the apical cells. The increased volume ofthe apical cells may have been due to either a longer periodbeing available for development as the rate of mitosis decreased,or to an unusually rapid rate of cell extension. The resultsindicated that cell division did not cease for lack of availablesugar nor as a result of failure to synthesize protein or nucleicacids. It is suggested that in the absence of boron divisionceases because abnormalities in the formation of the cell wallprevent the cell from becoming organized for mitosis. In particularthe hypothesis that boron is concerned with the formation of‘pectin’ from uridinediphosphate-D-glucose is examinedin the light of published evidence.     

Studies on the Growth in Culture of Plant Cells: IV. THE INITIATION OF DIVISION IN SUSPENSIONS OF STATIONARY-PHASE CELLS OF ACER PSEUDOPLATANUS L.

Techniques are described whereby a culture medium can be ‘conditioned‘by separation from a dense cell suspension either by a sinteror by a dialysis membrane. The enhanced growth-promoting activityof the conditioned, as compared with a new medium, is revealedby using a low density of cells (15 x 10³ or less cells perml) to initiate the test cultures from a stationary-phase suspension.The optimum pH of the conditioned medium is c6.4. To obtaina conditioned medium of high activity it is necessary to usean appropriate volume ratio of culture medium to conditioningcell suspension and to limit the conditioning period. Conditioningof the culture medium reduces by a factor of 10 (i.e. down toc. 1000 cells per ml) the minimum effective cell density neededfor self-sustaining growth. There therefore exists a population-dependentrequirement which is not met by the conditioned medium as nowprepared. The retention of the activity of the conditioned mediumin various situations has been studied as a preliminary to workon the chemical basis of conditioning.     

THE RATE OF GROWTH OF THE DAIRY COW : EXTRAUTERINE GROWTH IN WEIGHT.

The growth period of the Jersey and Holstein cows is made up of at least three cycles, two extrauterine cycles with maxima at about 5 and 20 months of age, and one intrauterine cycle, the maximum of which has not yet been determined. The equation of an autocatalytic monomolecular reaction was found to give very good results when applied to the cycle having its maximum at about 5 months of age. The values obtained from this equation when applied to the cycle having the maximum at about 20 months of age were higher than the observed values probably due to the retarding effect of pregnancy and lactation on growth.     

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.