Effect of 2,4-Dichlorophenoxyacetic Acid on Growth and on β-Glucan Synthetases of Peroxyacetyl Nitrate Pretreated Avena Coleoptile Sections

Coleoptile sections from Avena sativa L. were exposed to non-lethal concentrations of peroxyacetyl nitrate (PAN). The sections were then incubated in solutions of 50 mM glucose plus 2.5 mM potassium phosphate with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). Growth after 4 hours was measured. A corresponding series of experiments was carried out and the effect of the 2,4-D treatments on enzymes utilizing uridine diphosphate glucose (¹⁴C-glucose) to form glucolipid and β-glucans including cellulose was determined. Growth in the PAN-treated sections was inhibited less at optimal and superoptimal auxin levels than at low auxin levels. Glucolipid synthetase activity was only slightly inhibited by PAN pretreatment and was reduced by increasing levels of auxin. Responses of alkali-soluble glucan and cellulose synthetases were similar to growth in both control and PAN treated tissues. It was concluded that the earlier reported response of cell wall metabolism in vivo probably is due to effects on these enzyme levels.     

Inhibition of auxin-induced cell expansion in Jerusalem artichoke tuber slices by low concentrations of chloramphenicol

Summary D-threo chloramphenicol (CAP) at 5×10^-5 M, given continuously during a 24-hr aging period and subsequent post-age treatment with 2,4-dichlorophenoxyacetic acid (2,4-D)±kinetin markedly depressed cell expansion in Jerusalem artichoke (Helianthus tuberosus) tuber slices. Both the rate and total amount of expansion were reduced. An inhibitory effect of CAP could be detected at a concentration as low as 6.2×10^-6M with 2,4-D alone and 1.6×10^-6 M with 2,4-D+kinetin. CAP also inhibited if given with 2,4-D to unaged tissue, and partially inhibited growth of aged tissue when supplied only during or only after aging. Expansion was inhibited when IAA was used in place of 2,4-D. Growth of tissue slices free of detectable bacteria was depressed by CAP, eliminating a possible indirect action of the antibiotic through inhibition of beneficial bacteria. CAP also prevented appearance of pink and brown pigments which normally occur in association with auxin-treated tissues. L-threo CAP did not inhibit growth or pigment formation. Cell division in the tuber slices was not inhibited, and was possibly even stimulated, by D-threo CAP, even at a concentration of 2×10^-4 M. It is concluded that the use of CAP for bacterial control in plant cultures can be hazardous and needs careful checking. Presumably the inhibitory action of CAP results from inhibition of growth-dependant protein metabolism in mitochondria and/or plastids which occurs both during aging and post-aging growth. Partial suppression of metabolic changes during aging would maintain the tissue in a state favouring relatively high mitotic activity and slow growth in response to auxin.     

The Initiation and Maintenance of Vicia faba Tissue Cultures

Explants obtained by removing the radicle tip and the plumule from embryos of Vicia faba have been induced to form callus in culture. Of a range of agar-solidified culture media tested, only that of Schenk and Hildebrandt (1972) was consistently successful. Improved growth, measured as increasing fresh weight was obtained by increasing the nitrogen content of the medium, either as potassium nitrate or as ammonium nitrate. A kinetin concentration of 0.01 mg/1 (5 × 10⁻⁸M) and a 2,4-dichlorophenoxyacetic acid (2,4-D) concentration of 0.5 mg/1 (2.3 × 10⁻⁶M) allowed optimum initial callus growth. A 2,4-D concentration of 2.3 × 10⁻⁸M, while insufficient to induce callus formation was able to inhibit lateral root development which occurred from embryo explants cultured without added 2,4-D. Subcultured tissue grew well on media supplemented with casein hydrolysate or a mixture of the eight most common amino acids in casein hydrolysate. Growth in subcultures was inhibited by two other amino acid mixtures used by other workers for different species.     

Role of Auxin in Growth of Inhibitor-treated Oat Coleoptile Tissue

The role of auxin in the recovery of plant tissue from oxidant treatment was investigated. Treatment of oat coleoptile sections with concentrations of indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) optimal for normal growth, following pretreatment with moderately inhibiting levels of peroxyacetyl nitrate (PAN) immediately accelerated recovery of growth rate. In some cases inhibition was also less at supraoptimal values of auxin. Treatment of ozonepretreated tissue with IAA or 2,4-D enhanced inhibition at high levels of auxin and produced an optimal growth concentration level which was lower than for sections not given ozone pretreatment. Auxin treatment also reduced the degree of inhibition in fluoride and iodoacetamide-pretreated sections. Mechanisms by which auxin-induced recovery from inhibition may occur are discussed.     

Callus formation and plant regeneration from tubercles of Coryphantha elephantidens (Lem.) Lem.

Summary Callus cultures were established from pith tissue of Coryphantha elephantidens (Lem.) Lem. on Murashige and Skoog (MS) basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin. Highest shoot regeneration frequency was observed on a medium containing 6.9 μM kinetin and 2.3 μM 2,4-D under 30 μE m⁻² s⁻¹ light intensity with a 16-h photoperiod. Calluses retained organogenic potential throughout several passages of subculture (18 mo.). Shoots were rooted on MS medium without plant growth regulators. All (100%) plantlets transplanted to soil survived acclimatization. Regenerated plants showed good overall growth and were morphologically similar to the mother plants.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

Potassium influx and efflux of 2,4-D- and MCPA-treated rice plants

Summary A study was made of the effects of the herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methyl-phenoxyacetic acid) on ion uptake, leakage and growth of rice seedlings. Using isotopically-labelled solutions containing different concentrations of 2,4-D or MCPA, it was established that 10^–4 M 2,4-D or MCPA effectively inhibited potassium ion uptake, while K-ion leakage from the roots occurred only at 10^–3 M. The growth of the rice seedlings was markedly retarded even at low (10^–6 M) concentrations, and the roots and shoots showed different tolerances to the herbicide. At 10^–8 M herbicide, the effects were not injurious, but rather favourable. Reduction in root length by herbicides was not in accordance with dry-matter production.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Auxin Effects on the Aggregation and Heat Coagulability of Cytoplasmic Proteins and Lipoproteins

The kinetics of induction of heat stability of cytoplasmic proteins and lipoproteins by auxin (2,4,-D) were determined for basal sections of soybean hypocotyl. Maximum heat stabilization occurred after 4 h of tissue incubation with 10^-5M 2,4-D. The effect was less pronounced or absent with longer incubations. Membrane fractions sedimenting between 10,000 and 100,000 g and proteins of the 100,000 g supernatant were most affected. The auxin-induced protein aggregation response varied among experiments. With many tissue lots, the response was small or absent even though the tissue responded to the auxin uniformly by increased growth. The magnitude of response was proportional to the logarithm of auxin concentration but with low 2,4-D the portion of the homogenate protein coagulated by heat was increased and with supraoptimal concentrations it was decreased relative to the control. The smallest auxin-induced change in heat coagulability was observed at the auxin concentration nearest the optimum for growth. No direct correlation was found between the auxin-induced protein and lipo-protein aggregation phenomenon and total protein, chloroform-extractable lipid, residual lipid, growth or tissue deformability. Total sulfhydryl equivalent of the homogenates, however, did correlate with auxin effects on aggregation. This result, plus experiments where homogenates were exposed to oxidizing or reducing conditions, suggests that heat stabilization and associated protein aggregation phenomena are related to conversion of protein sulfhydryl to intramolecular disulfide bonds. No significance is attached to heat stabilization of cytoplasmic proteins as a requisite of auxin-induced growth.     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Genotypic Responses to Auxins in Tissue Cultures of Phaseolus

Responses of seven Phaseolus genotypes to the four auxins picloram, 2,4-D, NAA and 1AA, in tissue cultures, were examined. Callus growth was promoted by picloram and the range of effective concentrations for most genotypes was broad. The auxin 2,4-D also enhanced callus growth, but the range of optimal concentrations was markedly narrower than that of picloram. NAA when supplied at relatively high concentrations gave good growth. IAA was ineffective in supporting callus growth. The differences in 2,4-D concentrations required for optimal growth and the differential responses to low concentrations (0.04–1.25 μM) of picloram between several genotypes tested were large. These genotypic variations in auxin responses were repeatable and may thus reflect genetic differences.     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.     

The "Point of no Return" Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

The coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light - 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source). Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation. Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para-Fluorophenylalanine (p-FPhe) at 3.3 × 10^?2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released. It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p-FPhe no transitions were observed.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

Effect of cellulose synthesis inhibition on growth and the integration of xyloglucan into pea internode cell walls

2,6-Dichlorobenzonitrile (DCB, 100 μm) inhibited by 80 to 85% the incorporation of [³H]glucose into cellulose in stem segments of etiolated pea (Pisum sativum) seedlings. The inhibition lasted for at least 24 h. In the period 1 to 4 h after the excision of the segments, DCB did not influence elongation in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). However, during the period 1 to 24 h after excision, DCB enhanced endogenous and 2,4-D-stimulated elongation by 65 and 34%, respectively. DCB did not affect the incorporation of ³H from [³H]arabinose into xyloglucan, and did not change the ability of the [³H]xyloglucan formed in vivo to bind strongly to the cell wall. Therefore, at least 80 to 85% of newly synthesized cellulose was excess to the requirements for tight wall binding of newly synthesized xyloglucan. This conflicts with the hypothesis that xyloglucan is held in the cell wall solely by direct hydrogen bonding to the surfaces of cellulosic microfibrils.     

Uptake Kinetics of 2,4-Dichlorophenoxyacetate by Delftia acidovorans MC1 and Derivative Strains: Complex Characteristics in Response to pH and Growth Substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20–100 μM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 μM 2,4-D at either pH, and amounted to 15–20 nmol/min*mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min*mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Influence of silicon on cobalt, zinc, and magnesium in baker's yeast, Saccharomyces cerevisiae

Silicon (Si, as silicate) is involved in numerous important structure and function roles in a wide range of organisms, including man. Silicate availability influences metal concentrations within various cell and tissue types, but, as yet, clear mechanisms for such an influence have been discovered only within the diatoms and sponges. In this study, the influence of silicate on the intracellular accumulation of metals was investigated in baker's yeast (Saccharomyces cerevisiae). It was found that at concentrations up to 10 mM, silicate did not influence the growth rate of S. cerevisiae within a standard complete medium. However, an 11% growth inhibition was observed when silicate was present at 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown without added silicate (−Si) or with the addition of 10 mM silicate (+Si). Decreased amounts of Co (52%), Mn (35%), and Fe (20%) were found within +Si-grown yeast cultures as compared to −Si-grown ones, whereas increased amounts of Mo (56%) and Mg (38%) were found. The amounts of Zn and K were apparently unaffected by the presence of silicon. +Si enhanced the yeast growth rate for low-Zn²⁺ medium, but it decreased the growth rate under conditions of a low Mg²⁺ medium and did not alter the growth rates in high Zn²⁺ and Co²⁺ media. +Si doubled the uptake rate of Co²⁺ but did not influence that of Zn²⁺. We propose that a possible explanation for these results is that polysilicate formation at the cell wall changes the cell wall binding capacity for metal ions. The toxicity of silicate was compared to germanium (Ge, as GeO₂), a member of the same group of elements as Si (group 14). Hence, Si and Ge are chemically similar, but silicate starts to polymerize to oligomers above 5 mM, whereas Ge salts remain as monomers at such concentrations. Ge proved to be far more toxic to yeast than Si and no influence of Si on Ge toxicity was found. We propose that these results relate to differences in cellular uptake.     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A 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.