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An increasing fraction of volatile organic compounds (VOC) emissions come from the domestic use of solvents, contained within myriad commonplace consumer products. Emission rates are often poorly characterized and depend significantly on individual behavior and specific product formulation and usage. Time-concentration profiles of volatile organic compounds (VOCs) arising from the use of a representative selection of personal care products (PCPs) during showering are generated, and person-to-person variability in emissions calculated. A panel of 18 participants used a standardized set of products, dosages, and application times during showering in a controlled indoor bathroom setting. Proton transfer mass spectrometry was used to measure the in-room VOC evolution of limonene (representing the sum of monoterpenes), benzyl alcohol, and ethanol. The release of VOCs had reproducible patterns between users, but noticeable variations in absolute peak concentrations, despite identical amounts of material being used. The amounts of VOC emitted to air for one showering activity were as follows: limonene (1.77 mg ± 42%), benzyl alcohol (1.07 mg ± 41%), and ethanol (0.33 mg ± 78%). Real-world emissions to air were between 1.3 and 11 times lower than bottom-up estimates based on dynamic headspace measurements of product emissions rates, likely a result of PCPs being washed away before VOC evaporation could occur.

Volatile organic compound (VOC) emissions from personal care products (PCPs) contribute to poor indoor air quality. Exposure to indoor VOCs is typically determined through ambient concentration measurements; however, for some PCPs the proximity of use to the nose and mouth may lead to disproportionately large inhaled doses. In this paper, we quantify emission factors for six common PCP ingredient VOCs (ethanol, 2-propanol, benzyl alcohol, 1,3-butanediol, t-butyl alcohol, and the grouping of monoterpenes as limonene) from 16 facial day-moisturizers using headspace analysis and selected ion flow-tube mass spectrometry. A wide range of emissions rates were observed across the range of products tested (e.g., ethanol 3.3-6.9 × 102 µg s-1 g[product]-1 , limonene 1.3 × 10-1 -4.1 × 10-1 µg s-1 g[product]-1 ). We use a mannequin head with reconstructed nose and mouth airways to sample VOCs from facial application at typical respiration volumes. A single facial application of moisturizer can lead to a much larger inhaled VOC dose than would be inhaled from typical indoor ambient air over 24 h (e.g., limonene up to ~×16 greater via facial application, ethanol up to ~×300). Emissions from facially applied PCPs typically decayed to background concentrations over periods ranging from 5 to 150 min.

Volatile organic compounds (VOCs) emitted from personal care products (PCPs) can affect indoor air quality and outdoor air quality when ventilated. In this paper, we determine a set of simplified VOC species profiles and emission rates for a range of non‐aerosol PCPs. These have been constructed from individual vapor analysis from 36 products available in the UK, using equilibrium headspace analysis with selected‐ion flow‐tube mass spectrometry (SIFT‐MS). A simplified speciation profile is created based on the observations, comprising four alcohols, two cyclic volatile siloxanes, and monoterpenes (grouped as limonene). Estimates are made for individual unit‐of‐activity VOC emissions for dose‐usage of shampoos, shower gel, conditioner, liquid foundation, and moisturizer. We use these values as inputs to the INdoor air Detailed Chemical Model (INDCM) and compare results against real‐world case‐study experimental data. Activity‐based emissions are then scaled based on plausible usage patterns to estimate the potential scale of annual per‐person emissions for each product type (eg, 2 g limonene person−1 yr−1 from shower gels). Annual emissions from non‐aerosol PCPs for the UK are then calculated (decamethylcyclopentasiloxane 0.25 ktonne yr−1 and limonene 0.15 ktonne yr−1) and these compared with the UK National Atmospheric Emissions Inventory estimates for non‐aerosol cosmetics and toiletries.

Purpose: Limited drug penetration in solid tumors is a potential mechanism of resistance for many anticancer drugs. Taxanes represent a class of drugs that are currently undergoing a new round of development, but with little known of their ability to penetrate and distribute relative to blood vessels within solid tumors. Experimental Design: We assessed the tissue penetration of paclitaxel and docetaxel in HCT-116 tumor xenografts and in multilayered cell culture (MCC), a three-dimensional cell culture model of the tumor extravascular compartment. In xenografts, taxanes were mapped relative to blood vessels to obtain drug profiles as a function of distance from vasculature. For MCC, cultures were exposed to stirred drug reservoirs and taxanes measured as a function of depth into tissue. Results: Both taxanes exhibited limited penetration, with little drug reaching further than 100 μm into the tissue. Of the two, paclitaxel exhibited up to 2-fold greater penetration than docetaxel. Mapping tumor cell proliferation following treatment allowed the consequences of limited drug penetration to be assessed. In tumor xenografts where reduced drug exposure to cells far from vasculature is one of several factors influencing response to treatment, up to a 75% reduction in S-phase cells was achieved in cells nearest the vessels, but only 50% reduction was observed in the tissue 150 μm away. In MCC-based data, where the influence of reduced cell proliferation with depth into tissue was circumvented, a 5-fold (paclitaxel) and 10-fold (docetaxel) increase in reservoir drug concentration was required to produce a response in cells 150 μm into the tissue equivalent to that seen in cells directly exposed to the drug. Conclusion: These results indicate that limited distribution is an important mechanism of tumor resistance to taxanes.

Embryogenic masses (EMSes) of pistachio (Pistacia vera L.) were proliferated in liquid Murashige and Skoog (MS) medium without growth regulators. To determine the effects of benzylaminopurine (BAP), racemic (±) abscisic acid (ABA) and sucrose treatments during maturation on the subsequent germination and plantlet regeneration, clusters of mature somatic embryos were transferred from maturation medium onto the surface of 0.7% agar-solidified Murashige and Skoog medium. Neither germination nor plantlet development medium contained BAP or ABA. Germination studies were carried out using 80 somatic embryos at every combination of four sucrose concentrations, three maturation periods and either five concentrations of BAP or four of ABA, and the numbers germinating were recorded after four durations of culture. A similar experimental plan was used to study plantlet regeneration. The number of germinated somatic embryos increased markedly with duration of the culture on germination medium, and was influenced by the concentrations of BAP or ABA in the maturation medium; the concentration of sucrose in this medium had little influence. Plantlet regeneration also increased with culture duration and was reduced at the highest levels of BAP or ABA; with ABA, the probability of plantlet regeneration was lower for longer maturation periods. ABA and BAP have similar effects on somatic embryo germination (except at the highest levels used), but BAP is superior to ABA for promoting subsequent plantlet regeneration. Linear logistic models were used to investigate the significance of the treatments, and to estimate the optimum conditions for germination and plantlet regeneration.

Transformed hairy root cultures of chilli pepper (Capsicum frutescens cv. cayenne) containing the CaMV 35S promoter linked to the parsley PAL-2 cDNA were generated. These transformants showed increased PAL activity at early and late stages of culture. The morphology, colour and growth rate of these transformants were quite different from the control hairy roots. Measurements of cell dry weight, content of fibre and lignin-like material, suggested that the altered characteristics of the PAL transformants might be linked to lignification. HPLC analyses of phenolic compounds in transgenic roots revealed the accumulation of several substances that were not found in the controls. In response to the addition of salicylic acid or phenylalanine, lignification of the control hairy roots increased. However, the content of lignin-like substances in the PAL transformants was not increased by these treatments. These results indicate that lignification of the PAL transformants had reached a level that could not be further increased.

Summary Nicotine N -demethylation to nornicotine has been studied in cell-free preparations from cell suspension cultures of Nicotiana tabacum , in attempt to explore the mechanism of this enzymatic reaction. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity (2.8 × 10 −5 U) was present in the intermediate pellet whilst the maximum specific activity of 8.8 × 10 −5 U per mg protein appeared to be associated with the microsomal fraction. The addition of selected putative methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity. In addition, studies involving Sephadex G-25 gel filtration showed that molecules smaller than 5,000 MW are unlikely to be involved in this reaction. The current findings appear to support an oxidative nicotine N -demethylation and, together with the results from previous studies, provide some tentative evidence for the involvement of cytochrome P-450 in nicotine N -demethylation. The possible mechanism is also discussed.

This review attempts to present an overall strategy for the production of useful secondary metabolites by cultured plant cells. After consideration of the nature and utility of secondary metabolites and the possible role of these substances to the plant, the review focuses attention on the properties of the plant cells in culture and how the cell populations and their physical and chemical environment can be manipulated to encourage the synthesis and accumulation of secondary products. Finally, consideration is given to the involvement of genetic engineering in the production of cells to perform particular metabolic tasks and how these techniques might contribute to the development of a new strategy to enable the production of useful secondary metabolites on a commercial scale. CONTENTS Summary 553 I. Introduction 553 II. Development of a strategy 555 III. Concluding remarks 565 Acknowledgements 565 References 565.

The activity of the enzyme(s) catalysing the bioconversion of nicotine to nornicotine has been demonstrated, for the first time, in cell-free preparations from tobacco cell cultures. Using 14 C-assay, it has been shown that a maximal specific activity of ca 0.2 pkat mg protein −1 is present exclusively in the supernatant fraction after centrifugation at 8 800 g . The enzyme has a pH optimum between 9.0 and 9.5, and a temperature optimum between 25 and 30°, while the V max and the apparent K m are 7.6 × 10 −2 pkat and 7.4 μM of 14 C-nicotine, respectively. The decline in enzyme activity, following the removal of some endogenous co-factors and co-enzymes by dialysis of the crude enzyme preparation, can be restored and indeed enhanced by the addition of NADPH, suggesting that this enzyme may be NADPH dependent.

Radioactive feeding experiments, in which dl -[pyrrolidine-2′- 14 C]-nicotine was added to 10-day-old cultures, confirmed that the kinetic pattern of this N -demethylation was similar to that of non-radioactive nicotine, and that nornicotine, the major product, was produced intracellularly with a maximum percentage conversion of about 70%. The appearance of nornicotine paralleled the disappearance of the added nicotine, although small amounts of radioactive metabolites other than nornicotine were also observed. One of these metabolites was tentatively identified by GC-MS as N -formyl-3′-nornicotine which is probably a side-product rather than an intermediate in nicotine N -demethylation. Nornicotine produced from added (−)-nicotine by the cultures was determined using both polarimetry and chiral GC, and shown to be exclusively one enantiomer. This provided strong evidence that the mechanism of this reaction in cultured cells was unlikely to involve opening of the pyrrolidine ring and, therefore, differed from that previously hypothesized for the tobacco plant. The possible mechanism for this reaction is also discussed.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl−1 casein hydrolysate, 114 μM 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 μM benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 μM benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-14C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.

summary Examination of cryo-sections from mature rhizomes, immature rhizomes and adventitious roots of ginger, Zingiber officinale Roscoe, showed the presence of yellow-pigmented cells. There is a correlation between the number of pigrnenteci cells and the amount of [6]-gingerol, the main pungent principle in ginger, in these organs. It is also shown histochemically and microspectrophotometrically that the yellow cells contain, in addition to flavonoid-like compounds, possibly cureumin derivatives, phenolics and large amounts of lipid material. Low-temperature scanning electron microscopy also demonstrates the oil content of these cells. Therefore, it would appear that the site of accumulation of flavonoids, curcumin derivatives, phenolics including gingerol and the essential oils of this spice lies within the same cell type.

Two rapid techniques of column chromatography, flash chromatography and vacuum chromatography, together with solvent systems differing in polarity, have been used to separate the pungent principles (gingerols and shogaols) of an extract of ginger powder. Gingerols consist of a homologous series of aldols each containing a phenolic group which together with shogaols, showing similar structure, give the pungency of ginger. Thin-layer chromatographic analysis of the fractions collected from both chromatographic techniques showed that the main gingerol homologues are separated satisfactorily from the other compounds only using vacuum chromatography. These compounds were eluted with hexane-diethyl ether (30:70) and (20:80) respectively. The results obtained with flash chromatography, on the other hand, were not satisfactory.

A comparison of morphology, trichome type and duvane accumulation of tobacco shoot cultures originating from seedlings, callus and cell suspension cultures incubated on a range of cytokinin concentrations is presented. A method for the extraction and HPLC analysis of duvatrienediols from cultures is described. Duvatrienediols (DVTs) were not detected in callus or cell suspension cultures and were only detected in seedling-derived shoot cultures. Shoot development was dependent on explant type probably as a result of pre-exposure of callus and suspension cells to high auxin concentrations. Low cytokinin levels promoted shoot development whilst high cytokinin levels increased the number of shoots regenerated but retarded their development. DVT accumulation occurred only in 'normally' developed shoots with trichomes. Growth of shoot cultures in liquid medium was inhibitory to the accumulation of DVTs. Optimum conditions for the accumulation of DVTs were shown to be incubation of seedling-derived shoot cultures on media containing 10~6 M BAP solidified with agar. The yield was 120 /xg g"1 fr. wt. which is comparable to greenhouse-grown material. Both a- and /3-DVT were detected in a ratio of 5:1. The relationship between trichomes and DVT accumulation is discussed with reference to the roles of culture origin, cytokinin, trichome type and environmental effects.

summary Plant cell suspension cultures of Nicotiana tabacum cv. Wisconsin-38 accumulate nornicotine as the major alkaloid, unlike the parent plant where nicotine predominates. Nicotine was added to cultures of this cultivar, grown with differing degrees of dependence on photosynthesis for the provision of organic constituents, and also to cultures grown on medium that was optimised for alkaloid accumulation. Under optimal conditions nicotine was converted to nornicotine at an efficiency close to 100% over 10 d. Conversion appeared to he intracellular, with nicotine being taken up from the medium, demethylated to nornicotine and the product released. Illumination of cultures caused a slight decrease in conversion but, when cultures were partially photosynthetic, both growth and the degree of biotransformation were decreased by approximately 40%. As well as the four main tobacco alkaloids several minor alkaloids were detected in cultures after incubation with added nicotine.

The effect of light on alkaloid accumulation in a range of cell cultures of tobacco was determined. Cell suspension cultures of Nicotiana tabacum L. cv. Wisconsin-38 with differing degrees of photosynthetic activity, callus cultures of N. glauca Graham, root cultures of N. rustica L. and shoot cultures of N. tabacum were used. The alkaloid content of green illuminated cultures was greatly reduced compared with non-green cultures grown in the dark, but decreased accumulation did not correlate with increasing photosynthetic activity. The accumulation of all of the major alkaloids was affected, regardless of the species of tobacco used. Transfer of N. glauca callus from the dark into the light caused a decrease in alkaloid accumulation, while moving cultures from the light into the dark resulted in an increase in alkaloid content. In root cultures light caused a reduction in growth, which affected alkaloid synthesis. In shoot cultures there were only traces of alkaloid detectable, regardless of whether or not cultures were illuminated. Light appeared to cause a non-photosynthetic suppression of alkaloid accumulation in visibly undifferentiated cultures, and this effect was modified in visibly differentiated cultures.

This study determined neuron losses, mossy fiber sprouting, and interictal spike frequencies in adult rats following intrahippocampal kainic acid (KA) injections during postnatal (PN) development. KA (0.4 micrograms/0.2 microliters; n = 64) was injected into one hippocampus and saline into the contralateral side between PN 7 to 30 days. Animals were sacrificed 28 to 256 days later, along with age-matched naive animals (controls; n = 20). Hippocampi were studied for: (1) Fascia dentata granule cell, hilar, and CA3c neuron counts; (2) neo-Timm's stained supragranular mossy fiber sprouting; and (3) hippocampal and intracerebral interictal spike densities (n = 13). Mossy fiber sprouting was quantified as the gray value differences between the inner and outer molecular layer. Statistically significant results (p0.05) showed the following: (1) Compared to controls, CA3c and hilar neuron counts were reduced in KA-hippocampi with injections at PN 7-10 and PN 12-14 respectively and counts decreased with older PN injections. Granule cell densities on the KA-side and saline injected hippocampi were not reduced compared to controls. (2) In adult rats, supragranular mossy fiber sprouting was observed in 2 of 7 PN 7 injected animals. Compared to controls, increased gray value differences, indicating mossy fiber sprouting, were found on the KA-side beginning with injuries at PN 12-14 and increasing with older PN injections. On the saline-side only PN 30 animals showed minimal sprouting. (3) Mossy fiber sprouting progressively increased on the KA-side with longer survivals in rats injured after PN 15. Sprouting correlated positively with later PN injections and longer post-injection survival intervals, and not with reduced hilar or CA3c neuron counts. (4) On the KA-side, mossy fiber gray value differences correlated positively with in vivo intrahippocampal interictal spike densities. These results indicate that during postnatal rat development intrahippocampal kainate excitotoxicity can occur as early as PN 7 and increases with older ages at injection. This rat model reproduces many of the pathologic, behavioral, and electrophysiologic features of human mesial temporal lobe epilepsy, and supports the hypothesis that hippocampal sclerosis can be the consequence of focal injury during early postnatal development that progressively evolves into a pathologic and epileptic focus.

The present study examined the relationship between the patterns and densities of glutamate AMPA receptor sub-units GluR1 and GluR2/3 in the molecular layer of the fascia dentata and aberrant mossy fiber neoinnervation in human and kainate rat hippocampal epilepsy. Because AMPA sub-units modulate the fast glutamate synaptic transmission, we hypothesized that the AMPA receptor densities would be related to the glutamate-secreting mossy fibers, which could then contribute to seizure generation. In human hippocampal epilepsy, we found that the immunocytochemical labeling of GluR1 and GluR2/3 dendrites was positively related to the densities and spatial locations of the densest, aberrant neo-Timm stained supragranular mossy fibers. We used quantitative densitometry for the mossy fibers. However, the relatively faint and punctate immunocytochemical staining of the receptors did not allow true quantitative densitometry of the dendritic trees because in human epilepsy granule cell densities were decreased on average 50% of normal. Nevertheless, visual observations did confirm spatial relations between dense fascia dentata inner molecular layer mossy fibers and dense AMPA receptor staining. In the outer molecular layer, the mossy fibers were present only in the lower portion, were not densely-stained, and the AMPA receptors were only faintly-labeled. Nevertheless, outer molecular layer AMPA receptor densities were usually present more distally than were the mossy fibers. Experiments were done using intrahippocampal kainate epileptic rats to test the time courses for the changes in mossy fibers and AMPA receptors. The upregulation of inner and outer molecular layer AMPA receptors occurred maximally within 5 days post-kainate injection, prior to any mossy fiber supragranular ingrowth. One hundred and eighty days after ipsilateral kainate the AMPA receptors were increased bilaterally in the inner and outer molecular layers despite the fact that the contralateral aberrant supragranular mossy fibers were minor in comparison to the dense ipsilateral mossy fiber hyperinnervation. These results suggest that in hippocampal epilepsy AMPA receptor numbers increase throughout the length of the molecular layer dendrites; however the AMPA receptor densities are greater in rough relation to the greatest aberrant mossy fiber presynaptic inputs. Interestingly, the receptor upregulation precedes the mossy fiber ingrowth and may play a role in initiating axonal sprouting or in maintaining the aberrant mossy fiber synapses.

The accumulation of [6]gingerol and [6]shogaol (phenolic pungent principles of ginger) was much higher in culture systems of Zingiber officinale where morphological differentiation was apparent. Cultures grown on a callus-inducing medium also accumulated these metabolites but to a lesser extent. There is a positive relationship between product accumulation and morphological differentiation, although unorganised callus tissue also seems to possess the necessary biochemical machinery to produce and accumulate some phenolic pungent principles. In contrast to earlier studies with the intact plant, there was no positive correlation between the amount of [6]gingerol accumulated and the number of pigmented cells in either of the culture systems investigated.

Pieces of an embryogenic mass (EMS) induced in culture from immature fruits of pistachio, Pistacia vera L., were encapsulated into calcium alginate beads. Somatic embryos were also encapsulated individually into calcium alginate beads to produce synthetic seeds. The viability of the encapsulated EMS and somatic embryos was investigated immediately following encapsulation, and after storage for 60 days at 4°C. The encapsulated-stored EMS fragments recovered their original proliferative capacity after two months storage following two sub-cultures, but non-encapsulated-stored EMS failed to recover. The conversion frequency of synthetic seeds to seedling plants was 14% after storage for 60 days at 4°C, from which it may be concluded that encapsulation is a practical procedure for short-term storage of embryogenic pistachio tissue, and may be applicable to the preservation of desirable elite genotypes.

Octyl gallate is an antioxidant (European Community number 311). It is used as a preservation agent in a wide variety of foods and other non-dietary substances. We report a case of a 49-year-old female with a 10-year history of 'burning mouth' and clinical erythema of the tongue, who, after investigation, proved to be allergic to octyl gallate. Management with an exclusion diet proved effective in both controlling the burning sensation and resolving the erythema of the tongue.

In an attempt to determine the potential factors controlling the biosynthesis of the secondary metabolite capsaicin by immobilized cell cultures of the chilli pepper, Capsicum frutescens Mill, labelling techniques using the radioactive precursor [(14)C]phenylalanine have been employed. Following preincubation treatments with either capsaicin (the end-product of the pathway) or sinapic acid, [(14)C]phenylalanine was applied and the movement of the label through the pathway and its eventual fate was followed. Results have shown that capsaicin, through a feedback-inhibition mechanism, negatively influences its own synthesis. Furthermore, capsaicin synthesis in these cells is not controlled via the activity of the enzymes phenylalanine ammonia-lyase and cinnamate 4-hydroxylase which may determine the rate of entry of metabolites into the phenylpropanoid pathway. The importance of other sinks for phenylalanine derivatives, which may compete for capsaicin precursors, has also been investigated. Surprisingly, protein proved to be only a relatively minor sink for phenylalanine with the great majority of the label rapidly ending up in covalently bound phenolics in the cell wall. Attempts to prevent this by applying sinapic acid were only partially successful. The importance of these results in relation to the possible control mechanisms which operate to control secondary metabolite synthesis in vitro is discussed.

The carrot, Daucus carota, is a commonly used vegetable which has changed in appearance due to plant breeding programmes. The development of the carrot can be divided into a period of rapid increase in fresh weight, a stage of constant fresh weight and a stage of floral development and decrease in fresh weight. An analysis of the dietary fibre content of the carrot and its chemical composition has been made, using the varieties Altrincham, Chantenay, Nantes and Scarlet Perfection, at different developmental stages. Fibre was measured as prepared fibre, NDF, ADF, cellulose and lignin. No differences were found between varieties except those due to the different developmental rates of each variety. Within each variety the ADF content increased during late stage 2 and the increase was apparently due to the cellulose component of the ADF. Despite the differences, however, in carrot fresh weight between varieties and also within each variety there is no difference in any fibre component of carrot per 100 g fresh weight.

A different synthetic approach for the preparation of the cis-bromofluorobis(ethylenediamine) and cis-chlorofluorobis(ethylenediamine)chromi-um(II1) cations is described. The complex cations were isolated as iodide salts and in the case of the chlorofluoro complex a perchlorate salt was also isolated.

A novel technique is described for the immobilisation of plant cells. The method is simple and does not require the use of potentially toxic gels for the entrapment process. Liquid-suspended cells of two species, Capsicum frutescens Mill. and Daucus carota L. were found to invade, and were strongly retained in, polyurethane foam particles over a 21 day period. The viability of te immobilised cells was high (70–80% were alive after 21 days). On agitation of the loaded foam particles in fresh liquid medium, at least 95% of the cells remained immobilised after 3 or 4 days.

The accumulation of the phenylpropanoid precursors of capsaicin in suspended and immobilised cell cultures of C. frutescens has been studied and compared with accumulation in whole pepper fruit. The use of HPLC techniques has revealed that the phenolic precursors of capsaicin are present in chilli pepper cells at extremely low levels, irrespective of the source of tissue or the developmental state. Radioactive tracer studies have indicated that the majority of the phenolic derivatives of phenylalanine are ultimately bound to the insoluble fraction of the cells. Results from experiments where immobilised cell cultures were grown under conditions which enhance capsaicin yield would suggest that the ‘diversion’ of compounds into this bound fraction has a considerable influence upon capsaicin biosynthesis in this system.

SUMMARY The effect of auxins and a cytokinin on induction of roots in cultured axillary shoots of Pinus brutia Ten, has been tested. Auxin was crucial for root initiation and the rooting response varied according to the type and concentration of auxin applied. Both auxin and cytokinin and the interactions between them affected the quantity and quality of the induced roots. Aerated non-sterile tap water was an effective rooting medium, comparable to agar. After planting out into soil, some of the influences of auxin and cytokinin could still be seen after six months. However, roots developed normally in the soil, displaying dichotomous lateral branches. The results draw attention to the need for care in the choice and application of the medium for the initial induction of roots. Results of greenhouse trials indicated that the most vigorous plants were obtained via the axillary shoots.

The relationship between alkaloid content, growth rate and differentiation was investigated in seven solanaceous species in culture. Evidence was obtained for an inverse correlation between the growth phase on the one hand and alkaloid content and cell organization (aggregation) on the other. Linear phase and stationary phase cells of Datura innoxia Mill, incorporated DL-(l-14C)ornithine-HCl differentially into protein and alkaloids with proportionately more label found in alkaloids in the stationary phase cells. On transferring callus of D. innoxia from high auxin medium (containing 10~5 M 2,4-D) to low auxin medium (containing 10-6 M, 2,4-D), the alkaloid content dropped concomitant with a decrease in chlorophyll content and an increase in growth rate and friability. The nature of the interrelationships between growth, differentiation and accumulation are considered.

Dormant meristems of fascicles explanted from 10-year-old, field-grown trees of Pinus brutia Ten. were cultured in vitro. Browning of cultured fascicles was reduced by including 150 mg l(-1) sodium diethyldithiocarbamate (SDD) in a cytokinin-containing medium. The stage of development of fascicles when placed in culture affected both shoot-bud production and the degree of browning. Only fascicles at an advanced stage of development had a high rate of shoot-bud production. Fascicles cultured for 6 weeks in initiation medium containing 150 mg l(-1) SDD and then for 4 weeks in initiation medium containing 1 mg l(-1) insoluble polyvinylpolypyrrolidone showed the highest rate (23%) of shoot-bud development and the lowest rate (15%) of browning. Elongation of activated shoot buds was considerably enhanced by reducing the concentration of cytokinin in the culture medium. Only shoots >/= 20 mm in height were capable of producing further crops of buds and shoots. When treated with a combination of auxin and cytokinin, only 16% of the elongated shoots produced roots.

SUMMARY The intense colour of anthocyanins has made it possible to identify the individual cells accumulating these compounds within cultures of Catharanthus roseus. Furthermore, the amount of anthocyanin per cell could be measured directly by microdensitometry thus revealing the degree of heterogeneity within the cell populations at any interval during the growth cycle. In this way the contribution to overall product yield of individual cells in the culture was assessed. It has been found using this approach that although some inter-experiment variation occurs, intracellular anthocyanin concentration varies very little and is therefore likely to be controlled by a feedback inhibition mechanism. In general, attempts to enhance the mean intracellular concentration of the pigment through the manipulation of culture conditions have failed. A possible mechanism of metabolic control of intracellular anthocyanin accumulation is discussed. The proportion of cells accumulating anthocyanins, however, varied considerably in cultures grown under different conditions but nevertheless always remained low, never exceeding 20% of the total cell population. The cause of this poor response is unclear but it is suggested that a close metabolic interaction may exist between the cells in the culture to bring about this effect. The possible relevance of these observations to other culture systems accumulating secondary metabolites is discussed.

Summary A novel method is described in which the two halves of an explanted internode of Lycopersicon esculentum, Datura stramonium or Nicandra physaloides may be grafted together successfully in sterile culture. An absolute requirement for the formation of a successful graft is the application of indole-3-acetic acid (IAA at 0.2 to 2.0 mg l−1) to the apical end of the internode. The addition of kinetin (0.2 mg l−1) to the culture medium stimulated graft development but gibberellic acid (GA3 at 0−5 mg l−1) was inhibitory. The development of the graft as measured by an increase in mechanical strength and the formation of vascular connections was similar to that observed in whole plant grafts; however, fewer differentiated wound vessel members (WVMs) were detected. This technique should provide a powerful tool for the further study of graft development in compatible and incompatible combinations.

Plants of Datura stramonium (thorn-apple) were dissected into their component tissues and examined for the presence of the Datura lectin. This lectin was easily detected in seeds and in various parts of the flowers of adult plants. Traces were also found in green (emerged) cotyledons and roots of seedlings. The specific lectin activity in seeds contained within the fruits increased as the seeds matured. Mature seeds were homogenized in sucrose and separated by differential centrifugation into four fractions, three of which were clearly of distinct composition. Most of the lectin activity sedimented with the low-speed (cell-wall/protein-body) pellet, but a similar specific activity was recovered from the other fractions. However, if EDTA was included in the homogenization medium, three or four times more lectin activity was recovered in the soluble fraction. Immunofluorescent staining of formaldehyde-fixed sections showed that the lectin was localized in the cytoplasm, with little associated with cell walls. The possible relevance of these results to the function of the lectin in plant cells is discussed.

Important physical properties of dietary fibre include water holding capacity (WHC) and cation exchange capacity (CEC). In this investigation fibre prepared from different varieties of the same species, the carrot Daucus carota, have been sampled at different stages of development to determine whether these physical properties were dependent upon variety and the age of the plant. Results for WHC (g g−1 fibre) were dependent on the method of analysis. Differences found between varieties for WHC measured by centrifugation (mean 23.7 g, range 13.4–32.3) were not apparent when measured by filtration of a corresponding sample (mean 27.9 g, range 17.2–45.5). Differences between methods probably reflect structural differences in the fibre influenced by age and variety. Both methods showed carrot fibre to have a relatively high WHC. The CEC (meq g−1 fibre) was found to be similar between varieties but to increase with age within each variety, (mean 1.44 meq, range 1.07–2.42). The type of exchange did not change and the CEC 100 g−1 fresh carrot remained constant between varieties and also within each variety. The pK determined for each sample was between 4.42–5.30 which suggests pectins to be responsible for the CEC.

1. 1. Using the technique of density-labelling with deuterium oxide, evidence has been obtained for the de novo synthesis of glucose-6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADPH + 1-oxidoreductase, EC 1.1.1.49), during the culture of synchronously growing plant cells. 2. 2. The entire increase in enzyme activity during the early cell cycles in this material can be accounted for by the appearance of an enzyme species with increased bouyant density. 3. 3. A method is described for resolving overlapping distribution profiles after density centrifugation, which allows estimation of the amount of each species present at different times, and calculation of the loss of activity of the light species present from the start of culture. 4. 4. Loss of activity of glucose-6-phosphate dehydrogenase in normal growing conditions in the presence of 2,4-dichlorophenoxyacetic acid is very much faster than in conditions which do not lead to cell division: in the absence of 2,4-dichlorophenoxyacetic acid, or in the presence of the inhibitor of RNA synthesis, 6-methylpurine.

Summary The effects of malonate on the gaseous exchange and acid metabolism of mycelial suspensions of Aspergillus niger have been studied. Malonate penetrated into the cells of the fungus but did not exert its normal effect as a competitive inhibitor in the TCA cycle, even at high external concentrations. On the contrary it stimulated oxidative catabohsm. Some of the malonate which penetrated was consumed by the fungus and it is inferred that the remainder was stored within the mycelium away from malonate sensitive centres in the cytoplasm. Experiments performed with [2-14C] malonate have shown that part of the malonate molecule may be utilized in the synthesis of larger structural molecules of the mycelium. Possible pathways of malonate metabolism are discussed in the light of work carried out with other organisms.

The culture of plant tissues in an agitated liquid medium eliminates many of the disadvantages ascribed to the culture of tissues on agar. Movement of the tissue in relation to the nutrient medium facilitates gaseous exchange, removes any polarity of the tissue due to gravity, and eliminates nutrient gradients within the medium and at the surface of the cells. The incubation of a friable callus in a liquid nutrient medium agitated on a shaking machine, will eventually give a suspension of cells which are more amenable to experimental manipulation than callus grown on agar. The technique described here is one in which a callus of Daucus carota is suspended in a liquid nutrient medium and is subjected to a series of manipulations to ensure the production of an actively growing cell suspension consisting of single cells and very small aggregates of 2–15 cells.

The earliest successful sterile cultures of parts of higher plants were started from excised primary seedling roots. Root tips are attractive candidates because of their meristematic nature, availability, and the property of remaining relatively unperturbed after excision. In these early experiments the excised root tips were grown on a solid substratum from which the excised organs derived both nutrients and mechanical support. The media used were fairly simple in composition and contained, in addition to a natural extract, a carbon source, glucose or sucrose, and a mineral salts mixture consisting of the essential macro and micro elements. Although roots were subcultured with some success it was left to P. R. White to show that detached roots could be grown in aseptic culture for unlimited periods. Subsequently, in a series of investigations roots of pea and tomato have been cultivated in vitro and also used to study basic growth phenomena.

Capsaicin belongs to a group of phenolic compounds the capsaicinoids. These molecules, which are the end products of metabolism, are responsible for the hot taste in the mature fruits of the chilli pepper, Capsicum frutescens (Yeoman et al. 1980). They have a modest commercial value and at today’s prices one kilogram of the oleoresin would be worth about £450. Apart from the commercial implications there are certain features of capsaicin, its synthesis and accumulation, which form an attractive basis for those wishing to work with a system to explore the regulation of secondary product biosynthesis in general. For example, capsaicin, the predominant capsaicinoid in fruits, constitutes about 70% of the total with dihydrocapsaicin at about 28%. These capsaicinoids are only synthesized and accumulated in the maturing fruit at one particular stage of development, and have not been detected in other parts of the plant (Holden et al. 1987a). Indeed, all attempts to demonstrate capsaicin synthesis in leaves, stems or roots have so far failed (Holden et al. 1988a,b). From this it can be seen that a highly focussed approach is possible as the site and timing of capsaicin synthesis are known and the product is stable.

A different synthetic approach for the preparation of the cis-bromofluorobis(ethylenediamine) and cis-chlorofluorobis(ethylenediamine)chromi-um(II1) cations is described. The complex cations were isolated as iodide salts and in the case of the chlorofluoro complex a perchlorate salt was also isolated.

Cultures of higher plant cells can accumulate a variety of secondary metabolites such as polyphenols, alkaloids, steroids or certain pigments. Some of these secondary products can accumulate in the medium and may inhibit growth. This is particularly true of polyphenols. Other compounds, anthocyanins for instance, are not secreted by viable cells. Anthocyanins are a group of red, blue or violet pigments which occur in the vacuolar sap of plant cells, often in the epidermis of flowers and fruits. The products of hydrolysis of an anthocyanin are an aglycone bearing the generic name anthocyanidin and a glycoside unit, often glucose. Cultures which possess the ability to synthesise and accumulate pigments tend to lose this property during prolonged cultivation. This appears to be a phenomenon similar to the loss of morphogenetic capability with ageing of tissue cultures. Cells which can synthesise and accumulate pigments provide useful material in a practical course because the appearance and accumulation of the pigment can be monitored visually, as for example, with anthocyanin.

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g(-1) dry weight l(-1) medium compared with up to 30 μg g(-1)l(-1), respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-(14)C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [(14)C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.

Isolated plant protoplasts are cells from which the wall has been removed either mechanically or enzymically. The potential of cells without walls as tools to study cellular phenomena was appreciated by cell biologists as long ago as the end of the last century, but it was not until Cocking in 1960 showed that the wall of the constituent cells of the tomato fruit could be removed effectively by cellulase that sufficient material could be generated for experimental purposes. Much earlier, in 1936, Levitt had demonstrated that if cells from onion scale leaf epidermis are cut with a razor blade during strong plasmolysis in 20% sucrose and the cells were then transferred to a weak solution of sucrose, the protoplasts (protoplasm and vacuole) may actually swell enough to come completely out of the wall. Nowadays there are numerous reports of the successful isolation, culture and manipulation of protoplasts from a wide range of higher and lower plants. Leaves have proved to be the most amenable subjects for these procedures, particularly leaves of Nicotiana tabacum and Petunia hybrida.

It has been known for more than a century that the surfaces of cells bordering tissue air spaces are often ornamented with beads or elongated protuberances of material apparently containing pectin, as judged by their reactivity with ruthenium red (reviews by Kisser, 1928; Carlquist, 1956; Carr and Carr, 1975; Butterfield, Meylan and Exley, 1981). Pectic beads occur in all major groups of vascular plants and on cells from a wide variety of tissue types (Kisser, 1928). They have been observed by SEM in fern mesophyll by Carr and Carr, (1975) and in the ground parenchyma of Cocos nucifera stems by Butterfield et al., (1981) who reported that the beads stained with ruthenium red, although other tests for pectins were inconclusive. Davies and Lewis (1981) reported pectic projections on callus cells of Daucus carota consisting of more than one compartment, the outer one often containing oil droplets. They proposed that this oil may spread over the cell surface to provide a waterproof barrier, while Lipetz (1970) suggested it may be a prerequisite for wound healing. Nevertheless the function of pectic beads is still almost entirely obscure.