MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION

Investigations were carried out on the in vitro morphogenetic responses of inflorescence segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated’ the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.     

Role of phosphatidylinositol (PI) in ethanol production and ethanol tolerance by a high ethanol producing yeast

The effect of inositol addition on phospholipids, cell growth, ethanol production and ethanol tolerance in a high ethanol producing Saccharomyces sp were studied. Addition of inositol greatly influenced major phospholipid synthesis. With inositol in the fermentation medium, phosphatidylinositol (PI) content was increased, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were decreased. However, without inositol in the fermentation medium, PI content dropped down within 24 h, then increased, but was lower than in the presence of inositol. When yeast cells had a higher content of PI, they produced ethanol much more rapidly and tolerated higher concentrations of ethanol. During ethanol shock treatment at 18% (v/v) ethanol, yeast cells with a higher concentration of PI lost their viability much more slowly than those with a lower concentration of PI, indicating that the PI content in these yeast cells can play an important role in ethanol production and ethanol tolerance. Fatty acids and ergosterol were not responsible for high ethanol tolerance and high ethanol production in this yeast strain. Received 22 September 1998/ Accepted in revised form 20 December 1998     

A simplified medium for in vitro tracheary element differentiation in mesophyll suspension cultures from Zinnia elegans L.

A simplified medium has been developed for the differentiation of tracheary elements in suspension cultures of mesophyll cells of Zinnia elegans L. All inorganic salts contained in media used previously were retained in the simplified medium, but most were reduced in concentration. The only organic supplements required for optimum differentiation were thiamine and nicotinic acid, in addition to the plant growth regulators N6-benzylaminopurine and -naphthyleneacetic acid, and sucrose as a carbon source. Mannitol, an osmoticum, was necessary for rapid, synchronous differentiation. This simplified medium is particularly suitable for studies of the role of Ca²⁺ in tracheary element differentiation due to the elimination of myo-inositol, an intermediate in the phosphatidyl inositol signal transduction pathway and reduction in the concentrations of Mg²⁺ and Mn²⁺, which block calcium channels. It is also possible to eliminate EDTA from the medium, enabling studies using specific calcium chelators. Additional culture variables for the optimization of differentiation are discussed.Abbreviation TE tracheary element     

Agar and ammonium nitrate influence hyperhydricity,tissue nitrate and total nitrogen content of serviceberry (Amelanchier arborea) shoots in vitro

Shoot tip cultures of Amelanchier arborea Michx.f. were grown on Murashige & Skoog or Woody Plant (WP) medium containing 4.4 M benzyladenine and various concentrations of agar. Increases in agar concentration affected various culture growth variables, decreased culture hyperhydricity and increased tissue nitrate concentration. Additions of ammonium nitrate to cultures grown on WP medium containing 0.4% agar increased all growth variables measured except percent dry weight. Hyperhydricity and tissue nitrate concentration also increase in response to increasing ammonium nitrate in the medium. Since hyperhydricity was shown to be both positively and negatively correlated with increases in tissue nitrate content, it is unlikely that tissue nitrate level alone directly affects hyperhydricity.Abbreviations BA benzyladenine - MS Murashige & Skoog - WP Woody Plant     

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

Abstract: The psychotherapeutic action of Li⁺ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li⁺ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [³H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [³H]CDP-DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li⁺, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [³H]CDP-DAG was potentiated ～ 10-fold by Li⁺ and substantially reduced by coincubation with inositol. The formation of [³H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [³H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li⁺ potentiated carbachol-stimulated [³H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC₅₀ 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li⁺ to attenuate phosphoinositide resynthesis, as inferred from [³H]CDP-DAG accumulation. The results indicate that Li⁺ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.     

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomic tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.     

Regulation of MAL1+ gene expression encoding maltase in Schizosaccharomyces pombe by added inositol

In this study, the effects of inositol addition on maltase activity and expression of MAL1+ gene encoding maltase in Schizosaccharomyces pombe were investigated. The maximum specific maltase activity was observed, when the concentration of inositol reached 6.0 microg/ml in the synthetic medium containing 2.0% glucose. At 1.0 microg/ml inositol concentration, the maltase activity continuously decreased, as initial glucose concentration was higher than 0.1%. mRNA encoding maltase and phosphatidylinositol (PI) content were higher in the cells grown in the synthetic medium with 6.0 microg/ml of inositol and 2.0% glucose than those with 1.0 microg/ml of inositol. These results demonstrated that higher inositol concentration in the synthetic medium could derepress MAL1+ gene expression in S. pombe and PI might be involved in derepression of MAL1+ gene expression in S. pombe probably by PI-type signalling pathway.     

Changes in the composition of fatty acids and sterols of membrane lipids during induction and differentiation of Brassica napus (var. oleifera L.) callus

Changes in the membrane lipid and sterols content and composition were studied during induction and differentiation in callus cultures of Brassica napus var. oleifera. Callus induction was associated with an increase of DGDG content, significant changes in fatty acids composition of all lipid fractions and increased degree of lipid unsaturation. The membrane lipid composition of tissue at different degrees of differentiation was found to vary significantly, particularly two weeks after transfer of callus to regeneration medium. The main differences concerned the content and composition of galactolipids. Curiously in many cases, these differences declined during subsequent culture, in spite of the morphogenesis process which was in progress. Another result of differentiation was the change in free sterol composition: in shoot regenerating calli the content of stigmasterol had rose whereas the accumulation of campesterol decreased. Even though observed changes in membrane properties may not play a role in morphogenesis they are nevertheless useful as developmental markers and can be invaluable in understanding biochemical basis of morphogenesis.     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.     

Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Müll. Arg.)

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl₂ concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl₂ before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl₂ concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl₂ concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl₂ concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.     

GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two-medium sequence used to obtain whole plants. Callus of tetraploid clone S-4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4-D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4-D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4-D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4-D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract-inositol medium produced buds.     

Interstitial Ascorbate in Turtle Brain Is Modulated by Release and Extracellular Volume Change

Abstract: The isolated turtle cerebellum was used as a model system to study effects of depolarizing conditions on interstitial ascorbic acid concentration. The depolarizing stimulus was Leão's spreading depression, which is characterized by transient negative extracellular potentials, high potassium levels (20–60 μM), and local depression of neuronal activity. Interstitial concentrations of ascorbate (200–400 μM) and other electroactive species were monitored voltammetrically, using graphite fiber microelectrodes. Total tissue ascorbate (1,810 nmol/g tissue wet weight) was similar to mammalian levels and was several orders of magnitude higher than catecholamine and indoleamine content. During spreading depression, a large (up to 200 μM) increase in concentration of interstitial electroactive species was monitored. Use of Nafion-and ascorbate oxidase-coated electrodes and uricase confirmed that ascorbate was the only substance detected. Simultaneous monitoring of ascorbate, extracellular potential, and extracellular volume (using tetramethylammonium and ion-selective microelectrodes) indicated that (a) the ascorbate increase began with the decrease in extracellular volume during spreading depression, and (b) much of the increase was the result of extracellular volume decrease. In sucrose-substituted medium, in which volume changes are eliminated, a 50 μM increase in interstitial ascorbate, caused by release from intracellular stores, was also seen. The ascorbate concentration increase was prolonged in sucrose medium, suggesting that an uptake process involving sodium may further regulate interstitial ascorbate concentration.     

Inositol deficiency in yeast: metabolic,enzymatic and autoradiographic studies

The addition of inositol to starved cells of Saccharomyces cerevisiae NCYC 86 resulted in an initiation of growth. Inositol was incorporated into phosphatidylinositol and after a lag period RNA was the first macromolecule with a rate of synthesis departing from the rate observed in deprived cells. Pulse chase experiments showed that inositol was first incorporated into phosphatidylinositol and later into more polar lipids. Finally it appeared to be excreted into the surrounding medium. When S. cerevisiae NCYC 86 was grown in suboptimal concentrations of inositol (0,5 g/ml), alterations in the level of some membrane-bound enzymatic activities were detected; these might reflect structural modifications of the cellular membranes due to a different composition of phospholipids.High-resolution autoradiography showed that inositol was probably first incorporated into internal membranes and later transferred to the plasma membrane. Analytical experiments carried out with inositol-deprived cells showed that inositol was released into the surrounding medium in that case.The unbalanced growth detected in S. cerevisiae NCYC 86 under inositol deprivation might be due to an abnormal functioning of the cell membranes as a consequence of the deficiency in inositol-containing phospholipids.     

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.     

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K⁺] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K⁺].The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K⁺]₀. The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover.Increasing [K⁺]₀ produced a decline in [³H]inositol incorporation into PI in parallel with the increase in its labeling by ³³PO₄. This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected.Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [³H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.     

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Optimization of the Medium and Cultivation Conditions of Penicillium roquefortii f39 Producing the Diketopiperazine Alkaloid Roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5–2-fold. An increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and the amount of the inoculum. The use of sucrose in the inoculum medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

Effect of Light and Activated Charcoal on Tracheary Element Differentiation in Callus Cultures of Pinus Radiata D. Don

Light has been found to increase the proportion of tracheary elements differentiating in callus cultures derived from xylem-parenchyma of Pinus radiata D. Don grown on an induction medium containing activated charcoal but no phytohormones. The differentiation rate increased from 20% when callus was grown in darkness to 45% when callus was grown with a 16 h or 24 h photoperiod. When callus was grown with a 16 h photoperiod, tracheary elements were observed 2 days after transfer of callus to the induction medium, as compared to 5 days when callus was cultured in darkness. The differentiation rate was also influenced by the concentration of activated charcoal added to the induction medium, the optimum concentration being 5 g l⁻¹. Exclusion of activated charcoal from the induction medium decreased the differentiation rate to 2%. The activities of the lignin-related enzymes L-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase were significantly higher in cell cultures grown with a 16 h photoperiod as compared to when grown in darkness. The results show that light had a stimulating effect on tracheary element differentiation and the activities of lignin-related enzymes in P. radiata callus cultures. The new growth conditions markedly improve this cell culture system and make it particularly useful for functional gene testing and cell-wall analysis of in vitro grown tracheary elements of coniferous gymnosperms.