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Summary It is shown that an equal division in uninucleate microspores, giving two cells with similar staining nuclei, can initiate embryogenesis in anther cultures ofNicotiana sylvestris andN. tabacum var. Wisconsin 38 and evidence is presented that this pathway is quantitatively important and may be the only one leading to successful pollen embryos. Neither an initial asymmetric division in the uninucleate microspore nor the formation of a “suspensor” are essential to the development of embryo polarity.

Cell division was synchronised in 4-litre batch cultures of Acer pseudoplatanus L. by starvation and regrowth. Up to five consecutive cell cycles were observed in each culture. Mitosis and cytokinesis were synchronised within 0.2 cell cycles. Accumulation of extractable DNA was discontinuous and separate from cytokinesis. Correction for the degree of synchrony in the population gave: G1=13–37 h, S=15 h, G2=14–19 h and M=0.9–1.3 h. Thymidine kinase activity and [14C]thymidine incorporation were highest during S-phase. A peak of activity of aspartate transcarbamoylase occurred during G2. Peaks in succinate dehydrogenase activity and respiration rate were observed at the initiation of DNA synthesis and just prior to mitosis. The activity of glucose-6-phosphate dehydrogenase doubled in one step during the cell cycle. Total RNA and protein accumulated continuously through the cell cycle; the final rate being twice that observed initially.

The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth.

It is now possible, starting from isolated cells in culture, to regenerate higher plants, including many of importance in agriculture and horticulture. It is also possible to alter the genetic equipment of such cells and to induce the formation of fusion hybrids from them by enzymic removal of their cell walls. From such altered cells and such hybrids new plants can be generated. These technical achievements open up new possibilities in plant breeding and in the exploitation of the chemical potentialities of higher plant cells.

Development of haploid embryoids from the microspores of Atropa belladonna occurs with relatively high frequency when anthers are excised from buds in which the petals are shorter than the sepals (at this stage microspores are predominantly uninucleate) and cultured on a medium containing iron as the ferric salt of ethylenediamine-di-O-hydroxyphenylacetic acid (FeEDDHA). Additions of combinations of kinetin, auxin and casamino-acids to the culture medium induce callusing in both haploid and diploid tissues, lead to the origin of embryoids from somatic tissues of the anther and should be avoided. Simple techniques for the maintenance of haploid clones are described.

Embryoids are initiated from single highly cytoplasmic cells of the stem epidermis of plantlets of Ranunculus sceleratus L. derived from callus cultures. The embryoids show embryological development of the Crucifer type except that the suspensor is reduced or suppressed. The cells of the young embryoids have a large central nucleus surrounded by small vacuoles, a cytoplasm rich in free ribosomes or ribosomes in small polysomes, prominent amyloplastids and abundant spherosomes. These cells also frequently contain multivesicular and myelin bodies. The spherosomes are observed in various stages of development; they are typically arranged in a layer below the cell wall, the space between the spherosomes and the wall being rich in spherical vesicles and microtubules. As embryoid development proceeds the plastids show internal differentiation; globular centres and, surrounding them, electron-transparent droplets are associated with the origin of the thylakoid membranes. The walls of the embryogenic cells are initially rich in plasmodesmata but as the embryoid develops cytoplasmic continuity with the embedding tissue is severed and the embryoid is clearly delineated. The embryology and fine structure of the embryoid cells is compared with that of zygotic embryos of Capsella and Ranunculus.

Sections of callus of Ranunculus sceleratus reveal its organization into cellular aggregates, the superficial cells of which are highly cytoplasmic and the inner cells of which are larger, have less-dense cytoplasm and are more highly vacuolated. Expansion and ultimate death of some of the internal cells leads to cell separation and break up of the aggregates. Many of the superficial cells are involved in the initiation of embryoids in the callus and they correspond in structure with the embryogenic cells of the seedling stem epidermis. The embryoids are retarded in their development in presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and embryoid development becomes more rapid and more prolific when cultures are transferred to a medium without 2,4-D. The similarity between the embryogenic cells of the callus cultures and those of the seedling stem epidermis extends to cell size, nuclear size, degree of vacuolation, abundance of ribosomes and mitochondria, presence of amyloplasts and prominence of spherosomes. The various forms of spherosome are described and their possible function discussed. Amyloplasts differentiating into chloroplasts are observed in the more advanced embryoids. There is evidence that embryoids can arise from single cells but it is uncertain whether all are of single-cell origin. The embryogenic cells are in protoplasmic continuity with surrounding cells when they embark upon embryogenesis. Some of the superficial cells, also clearly undergoing active division, are of rather different structure; characteristically their nuclei show a high degree of chromatin condensation and their cytoplasm contains bundles of fibrous material. It is suggested that these cells do not function directly in embryogenesis. The internal cells of the aggregates have a low density of ribosomes and very few ER profiles or normal mitochondria. Extremely elongated mitochondrial structures following the outline of the nucleus are observed in these cells. An unidentified structure is frequently observed in cells in which cytoplasmic disorganization appears to be occurring.

Summary When suspensions of sycamore cells are cultured in a synthetic medium containing 1.0 mg/l 2,4-D and 0.25 mg/l kinetin, maximum cell yield is obtained with an initial concentration of 6 per cent sucrose. There is a progressive increase in dry weight per cell, decline in extractive-free weight as a percentage of cell dry weight and increase in lignin content per cell as the initial sucrose concentration is increased from 1 per cent to 15 per cent. The percentage of lignin in the extractive-free cell residue is further enhanced by increasing the level of 2,4-D to 10 mg/l or by growing the cells in an auxin-free medium containing 10 mg/l kinetin.

1Detailed knowledge of the growth requirements and growth responses of excised roots is available for only a few species, of which tomato (Lycopersicum esculentum)is the outstanding example.2Excised tomato roots have absolute requirements for thiamine and pyridoxine. The growth of some strains is further enhanced by niacin. The view is advanced that these requirements arise because the vitamins are synthesized in the roots at an inadequate rate rather than from a complete loss of synthetic ability. By use of possible precursors and analogues knowledge has been obtained relating to pathways of vitamin biosynthesis, the specificity of the vitamins and the ‘sparing action’ on vitamin requirements of certain metabolites.3Excised tomato roots can assimilate nitrate nitrogen. Recently the development of techniques for the adjustment and stabilization of the pH of the culture medium and the use of a stable ferric ethylenediamine tetra‐acetate have made possible growth on ammonium nitrogen.

MANY plant tissue cultures change in growth rate, chromosome cytology and morphogenic potential during repeated subculture1–3. Controlled freezing and low temperature storage of cultured plant cells might enable the characters of newly initiated cultures to be preserved. Cell preservation at the temperature of liquid N2(−196° C) has been successful with animal cells4and preliminary work with plant cells has shown the promise of this approach5–7. Latta has reported regrowth of cultures of carrot (Daucus carota L.) following freezing for up to 2 months at −40° C or at −196° C but did not assess the percentage of cells surviving6.

Anthers of Nicotiana tabacum produce ethylene when cultured for plantlet production. The rate is at a maximum 1–2 weeks after the onset of culture. Charcoal in the medium increases the proportion of androgenic anthers in N. tabacum and several other Nicotiana species. The level of ethylene in culture vessels is reduced by charcoal. However, complete removal of ethylene does not significantly alter the incidence of androgenesis, nor does continuous flushing of cultures with air. It is concluded that although charcoal reduces ethylene in the gas phase of the cultures its effect on androgenesis is exerted through some other mechanism.

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batch cultures of sycamore (AcerpseudoplatanusL.) caused growth cessation after an initial period of exponential growth. The presence of white light reduced the amount of growth after 2,4-D withdrawal. Growth cessation, in the absence of 2,4-D, was accompanied by the accumulation of the free amino acid, serine. This accumulation began before the cessation of growth and was rapidly reversed by the re-addition of 2,4-D.

A technique is described for the isolation of purified nuclei from suspension culture cells of Acer pseudoplatanus. This involves a grinding medium containing 70% (v/v) glycerol, 1 mM Mg2+, 2 mM Ca2+, and Tris buffer at pH 7.8, prestorage and disruption of the cells at −20 °C in a Potter-Elvehjem homogenizer, and purification by filtration and centrifugation in the presence of Triton X-100. The nuclear yield is c. 25% as assessed by nuclear count or DNA estimation and the nuclei are active in the RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H2SO4 and precipitated by ethanol, 113 μg histone is obtained per μg nuclear DNA and the histone fraction contains 22% basic amino acids and has a lysine: arginine ratio of 2.6. Acid-urea gel electrophoresis shows the presence of five major histones (H1, H2A, H2B, H3, and H4 in sequence from anode to cathode) having respectively molecular weights of 24 500, 13 500, 13 300, 12 800, and 11 000. There is very good correspondence between calf thymus histones H3 (reduced form) and H4 and two of these Acer histones. The other Acer histones differ from the calf thymus histones H1, H2A, H2B in molecular weight but can be provisionally equated with these by a newly developed differential staining reaction. Calf thymus histone H2A appears to be less rich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (14C)lysine and [3H]tryptophan is in favour of the cytoplasmic synthesis of the histones.

Pollen isolated from anthers of a Burley cultivar of N. tabacum by an anther homogenization procedure failed in culture to retain viability and produce plantlets even when isolation was preceded by 14 d of anther preculture. By surgical manipulation of anthers prior to their culture it was shown that pollen viability was very sensitive to injury caused to the anther wall. Plantlets could be obtained however by culture of anthers in which one or both pollen sacs had been carefully opened along the dehisence line (DL). The somewhat lower yield of plantlets from such anthers compared to that from intact anthers is considered to result not from any change in the atmospheric environment of the pollen as a result of opening the pollen sac but from the inability in every case to keep the incisions strictly along the dehiscence line. Pollen isolated from 14 d precultured anthers by this DL technique was able to yield plantlets when cultured in a simple defined medium. However DL-isolated grains from 4 d precultured anthers failed to retain viability in culture. It is concluded that pollen of this cultivar suffers severe injury when isolated by the homogenization method and that this can be overcome by the DL technique. However conditions of culture have not been established which reproduce for this cultivar the nutritional conditions operating within the anther during its early period in culture and which are essential to plantlet formation.

Growth of C. roseus cell suspension culture was defined in terms of dry weight, cell number, mitotic index, and packed cell volume. Removal of major nutrients from the medium was monitored as a function of culture growth. Phosphate and sucrose were the only macronutrients completely exhausted. Utilization of these two nutrients occurred parallel with increments in dry weight and cell number. Increasing the nutrient medium levels of sucrose and phosphate prolonged growth of this culture; lag and exponential phases were extended; cell number and dry weight yield were doubled. Dry weight assimilation was enhanced by increasing the nutrient medium level of sucrose, whereas increments in cell number were related to phosphate level. Two alkaloid fractions (fractions 1 and 2) were identified in this cell line. Fraction 2 alkaloid level declined as the nutrient medium supply of nitrogen was depleted.

Techniques have been established which give reproducible yields of total and of free and bound polysoines from cultured sycamore cells liarvested at intervals throughout the cycle of growth followed in batch culture. High levels of polysoines build up in the cells during lag phase and persist into early exponential growth. Later in the growth cycle, although the ribosomal material per unit volume of culture continues to rise, the content per cell of ribosomes and polysomes progressively declines until the cells enter the stationary phase. There is also a characteristic pattern of change in the relative proportions of free and bound polysomes throughout the growth cycle of the cultures. Both fractions contain different but significant levels of ribosomal subunits and monomers. The RNAs released from the free polysoines had a greater amount of poly(A) sequences than that from the bound polysomes. These findings are discussed in relation to the changing metabolic activities of the cells which occur during their progress through batch culture.

Three methods of cell cycle analysis, involving the use of tritiated thymidine, have been applied to asynchronously dividing suspension cultures of sycamore. Conditions for an effective chase of unlabelled thymidine were established from a study of the kinetics of entry and incorporation of tritiated thymidine into the cells. The levels of thymidine used did not affect the rate of cell division or the duration of the phases of the cell cycle. The analyses of the cell cycle based upon pulse labelling, continuous labelling, and a combination of densitometry and autoradiography were in good agreement and showed that the phases S (mean 7.0 h), G2 (mean 8.5 h) and mitosis (mean 3.0 h), were of relatively constant duration, whereas G1 was of variable duration. No relation between nuclear DNA content and mitotic-cycle time or the duration of S-phase could be inferred from the data presented.

[2-14C]-uridine is rapidly taken up by sycamore cells in suspension culture. A proportion of the radioactivity enters RNA without measurable delay, whilst the remainder equilibrates with a large pool of phosphorylated compounds, the major radioactive component of which is 5'-UMP. Both the uracil and cytosine residues of RNA receive label from [14C]-uridine and, when the cells are supplied with high concentrations of uridine, these bases are derived almost exclusively from the nucleoside. [14C]-uridine is incorporated into RNA at all stages of the growth 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-term labelling experiments also indicate turnover of RNA during the phase of active cell division and suggest that a large proportion of 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 external uridine concentration of 10−6M to 75 per cent at 10−3 M. Despite this, nucleic acids are the only macromolecules that receive a significant amount of radioactivity from [2-14]C-uridine.

Indolylacetylaspartic acid (IAAsp) and possibly indolylacetylglutamic acid (IAG) are formed by exposure of excised tomato roots to IAA. Little ‘free’ IAA accumulates in the tissue. An unidentified substance reacting pink with nitricnitrite reagent is also formed. These substances are metabolized when IAA-treated roots are transferred to auxin-free medium. IAAsp and IAA are similarly inhibitory to the growth of excised tomato roots. Excised tomato roots do not interconvert IAA and IAN. IAN-feeding leads to IAN accumulation and the appearance of indolylcarboxylic acid (ICA); transference to auxin-free medium causes a decline in the IAN activity but the ICA spot persists. The inhibitory activity of IAN is not due to its conversion to ICA. Excised tomato and wheat roots respond very differently to external l-tryptophane but in neither case is there evidence of the conversion of tryptophane to ethyl acetate-soluble auxins.

WHITE1has stated that secondary thickening does not occur in excised root cultures. Some of our cultures of tomato (‘Best of All’) which had been kept without transfer for five to six months were recently submitted to anatomical investigation, and were found to have developed a considerable amount of secondary xylem, though the occurrence of properly differentiated secondary phloem has not yet been demonstrated. The nature and extent of the secondary xylem can be illustrated by reference to two examples.

Aspects of the carbohydrate and nitrogen metabolism of Acer pseudcplatanus, L cell suspension cultures grown on a synthetic medium containing 2 per cent glucose and 1.0 mg/l 2,4-dichlorophenoxyacetic acid and kinetin either at 0.25 mg/l (low kinetin) or at 2.5 mg/l (high kinetin) are described. High kinetin inhibits growth as 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 cells contain only a trace amount of fructose and their rate of O2 uptake can be raised to that of the low kinetin cells by a period of fructose feeding. The O2 uptake of both kinds of cell is sensitive to malonate but the stimulation of O2 uptake induced by bis(hexafiuoroacetonyl)-acetone (‘1799’) at 0.2 mM is much less with the high-kinetin than the low-kinetin cells. The enzymes phosphoglucoseiseomerase and glucose-6-phosphate dehydrogenase are much less active in the high-kinetin cells. Mitochondria isolated from both kinds of cells show good respiratory control although slightly lower values for QO2(N), ADP/O ratio and control ratio are recorded with mitochondria from the high kinetin cells. Kinetin at 2.5 mg/l slightly reduces the ADP/O ratio of isolated mitochondria but at 4.0 mg/l their response to ADP is completely suppressed. Extracellular hemicellulose formed in presence of high kinetin has a reduced content of galactose and xylose and an increased content of glucose. These observations indicate that the inhibition of respiration by high kinetin is mainly due to suppression of glucose conversion to other sugars rather than to inhibition of glycolysis or terminal respiration. High kinetin decreases the rate of protein but not of amino-acid synthesis. Suppression of the synthesis of particular proteins may be an important factor responsible for the reduced cell yield of the cultures in presence of high kinetin. The significance of these observations to our understanding of the critical metabolic effects of cytokinina is discussed. Acer pseudoplatanus cells release amino acids into their culture medium early in the period of batch culture and largely reabsorb them as incubation proceeds.

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

Extensive ridge-like structures lining the outer walls of cells at the surface of cell aggregates and less elaborate thickenings on the walls between adjacent cells are reported and compared with the wall thickenings observed in ‘transfer cells’. Mitochondria with an atypical crista are described. Plastids in cells cultured in the standard synthetic medium contain poorly developed internal lamellae and function as amyloplasts during the growth cycle. Their differentiation into chloroplasts is induced by replacing the 2,4-D by NAA in the medium. A structure is proposed for the crystalloid core of the microbodies, which are a prominent feature of stationary phase cells.

A versatile large-scale batch culture unit has been developed for the growth of plant cell suspension cultures. This unit has been modified to permit of intermittent or continuous renewal of culture medium and, in a modified form, incorporated into open continuous culture systems of the chemostat and turbidostat type. A fully automatic culture sampler has been incorporated into the basic culture unit. The culture systems described have been successfully operated using a cell suspension derived from Acer pseudoplatanus and results are presented demonstrating synchronous growth in batch culture, prolonged logarithmic increasse in cell number under conditions of high aeration and culture medium renewal, and steady states of growth resulting from automatic regulation of the optical density of the cell suspension and from fixed rates of displacement of cell suspension by new medium. The potentialities of the culture systems are discussed in the light of the experimental results presented.

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.

Inhibitory concentrations of 3-indolylacetonitrile (IAN) cause, in cultured excised tomato roots, a marked decrease in the rate of cell division at the apical meristem but only a slight reduction in the lengths of mature exodermal and cortical cells. The reduced rate of cell division is associated with a decrease in the. number of meristematic cells at the root apex. By contrast, 3-indolylacetic acid (IAA) causes marked reduction in the lengths of mature cortical cells but does not markedly reduce cell-division rate at the apical meristem. Various lines of evidence indicate that both IAA and IAN cause a relative increase in the number of longitudinal and a decrease in the number of transverse division walls in the meristematic zone of the root apex. Partial inhibition of the linear growth of excised tomato roots by IAA and IAN is accompanied by increases in root and stelar diameters. These increases result from radial enlargement of the cortical cells and increase in the number of stelar cells in the transverse section. The enlarged steles contain an increased number of lignified xylem elements, but only with the most inhibitory concentration of IAN (10−4g./ml.) is there evidence of the development of secondary xylem. Both auxins increase significantly the xylem vessel unit length.

Extracts of excised tomato roots prepared with cold aqueous methanol have been partitioned between ethyl acetate and water and chromatograms of the two fractions bioassayed by an Avena coleoptile straight growth test and sprayed with various reagents including those giving colour reactions with indole compounds. The greater part of the auxin activity is in the aqueous fraction and chromatograms of this fraction give positive indole reactions. The aqueous fraction chromatographed with isopropanol/ammonia/water shows two zones of growth-promoting activity. The zone of lower Rf contains tryptophane and unidentified ninhydrin-positive material. Tryptophane accounts for the activity of the Ehrlich-positive region of this zone. The activity of the zone of higher Rf is associated with ninhydrin-positive material; this activity can, by use of other solvents, be distinguished in Rf from that of the common amino-acids which would occur in this zone on the primary chromatograms. Chromatograms of the aqueous fraction also show the presence of urea and of a phenol, neither of which are associated with the zones of growth-promoting activity. The ethyl acetate fraction, and particularly the acid fraction of this, contains growth-promoting activity which, in a range of solvents, always corresponds in Rf to IAA. The acid fraction contains a growth inhibitor corresponding in Rf to the β-inhibitor of other workers. A zone of growth-promoting activity near the solvent front on isopropanol/ammonia/water chromatograms contains more than one active component, but IAN appears to be absent.

I-naphthylacetic acid (NAA) enhances main axis extension and lateral initiation of excised tomato roots cultured in 0.5 per cent sucrose medium. NAA has a qualitatively similar effect upon the growth of root tips cultured in glucose medium or transferred after 2 days' previous growth in standard (1.5 per cent sucrose) medium to a glucose medium containing NAA.NAA does not raise the growth-rate of roots cultured in glucose medium to that occurring in presence of sucrose as carbon source. The enhanced growth resulting from addition of NAA to glucose medium is not dependent upon enhanced glucose uptake.The unique role of sucrose in the growth of excised tomato roots is discussed in the light of the above observations and of studies on the ability of glucose to maintain the growth of excised roots previously established by a period of culture in sucrose medium.

A technique has been developed for the electron microscope study of the free cells and small cell aggregates of suspension cultures of Acer pseudoplatanus, L. Changes in fine structure have been followed during the growth of a batch culture over 24 days, covering the lag phase, the phase of exponential growth, and the stationary phase to a condition where the cells show evidence of senescence. During the lag phase there is a massive synthesis of new cytoplasm and an increase in the number of mitochondria and ribosomes. By the point of transition to the phase of exponential growth many of the ribosomes are either attached to the ER membranes or are organized in spherical or spiral clusters. Multivesicular bodies are frequently observed. The development of the cell plate can be followed in some detail at this stage. As the rate of cell division decreases and cell enlargement begins the cytoplasm comes 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 15th day of culture, the old cell walls show characteristic changes and are frequently ruptured. Intra-cytoplasmic vacuoles appear and then with the continuation of culture disappear as the cytoplasmic layer approaches its minimum thickness. Nuclei show invaginations and these often contain characteristic ‘aged’ mitochondria.

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

Techniques are described whereby a culture medium can be ‘conditioned‘ by separation from a dense cell suspension either by a sinter or by a dialysis membrane. The enhanced growth-promoting activity of the conditioned, as compared with a new medium, is revealed by using a low density of cells (15 × 103 or less cells per ml) to initiate the test cultures from a stationary-phase suspension. The optimum pH of the conditioned medium is c6.4. To obtain a conditioned medium of high activity it is necessary to use an appropriate volume ratio of culture medium to conditioning cell suspension and to limit the conditioning period. Conditioning of the culture medium reduces by a factor of 10 (i.e. down to c. 1000 cells per ml) the minimum effective cell density needed for self-sustaining growth. There therefore exists a population-dependent requirement which is not met by the conditioned medium as now prepared. The retention of the activity of the conditioned medium in various situations has been studied as a preliminary to work on the chemical basis of conditioning.