The Effect of {alpha}-Naphthalene Acetic Acid (NAA) and Benzylaminopurine (BAP) on the Accumulation of Volatile Oil Components in Cell Cultures of Tarragon (Artemisia dracunculus)

Disorganized cells of A. dracunculus have been demonstratedto be competent to synthesize the four phenylpropene compoundscharacteristic of tarragon essential oil. Allylanisole and methyleugenol, the major components, are accumulated at particularlylow levels and are inhibited largely by the presence of exogenousNAA. Volatilization, active metabolism, and autotoxicity ofthese compounds are involved in limiting their accumulation.The data reported here suggest that both limited expressionof specific synthetic pathways, and the absence of suitable(inert) accumulation sites can restrict accumulation of phenylpropenesin disorganized cultures of A. dracunculus. Key words: Artemisia dracunculus, tarragon, tissue culture, essential oil, phenylpropenes, allylanisole, methyl eugenol     

Effective Cytochrome P450 (CYP) Inhibitors Isolated from Tarragon (Artemisia dracunculus)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC₅₀ values of 10.0 ± 1.3 µM for compound 1 and 3.3 ± 0.2 µM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.     

Experimental and Analytical Studies of Pteridophytes: XXI. Sterile cultures of an abnormal growth from the gametophyte of bracken

The isolation and growth in sterile culture through sixteenpassages, each of 4 to 6 weeks' duration, of an abnormal growthisolated from a gametophyte of Pteridium aquilinum var. latiusculumis reported, and the nutritional requirements described. The cultures consist of green, photosynthetic, predominantlyfilamentous, but coherent, masses. Prothalli were regeneratedon five occasions during the first two passages only. Outgrowthsof whitish or pale green parenchymatoua tissue occur. Thesepossess a central strand of elongated cells, some of which becomedifferentiated into a core of tracheides. Prothalli of the strain in culture are haploid. When examinedsoon after the initial explantation, the filamentous parts ofthe cultures were diploid, but there has been a subsequent increasein the number of chromosomes, and each cell is now irregularlyaneuploid, with a chromosome number between triploid and tetraploid.     

Purine Alkaloid Production and Accumulation in Cocoa Callus and Suspension Cultures

Cocoa callus and suspension cultures were found to produce caffeine,theobromine, and theophylline which are typical of the purinealkaloids found in cocoa beans. Production of these purine alkaloidswas monitored in callus cultures for over 2 years and shownto stabilize at concentrations of about 10% those found in vivo.Caffeine and theobromine were produced concomitant with logphase growth of the cultures whilst theophylline productionreached a maximum during stationary phase, reflecting the possiblerole of the latter as a catabolite of caffeine. The effectsof choice of cytokinin, explant tissue, cocoa type, light conditionsand time in culture on purine alkaloid production by callushave been examined. Purine alkaloid production by cocoa suspensioncultures has also been examined and these cultures were shownto be less productive and more variable than callus cultures.The results demonstrate that cocoa tissue cultures can be usefulfor studying secondary metabolism in vitro. Key words: Theobroma cacao, caffeine, theobromine, tissue culture, secondary metabolism     

Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography

Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-µm vertical projections that we term "pegs," to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (P < 0.001) and a concomitant 60% decrease (P < 0.01) in cyclin D1 protein levels, suggesting a G₁/S₁ cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 µM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-µm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo. tissue engineering; cell culture; cell cycle     

Analysis of the Contents of Oil Cells in Tasmannia lanceolata(Poir.) A. C. Smith (Winteraceae)

The simple, robust oil cells found in leaves of Tasmannia lanceolata(Poir.)A. C. Smith (Winteraceae) are shown to contain a preponderanceof the bioactive compound polygodial using both direct samplingand GCMS analysis of cell contents and Fourier Transform infraredspectroscopy. The implications of this for comparison of plantmaterial on the basis of extracts and essential oils are discussed.Copyright 2000 Annals of Botany Company Tasmannia lanceolata, Winteraceae, polygodial, direct sampling, FTIR spectrometry, oil cell, idioblast, antifeedant     

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02?mg/g DW in comparison with the nontransformed roots (up to 0.687?mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.     

Basal Plate Tissue in Narcissus Bulbs and in Shoot Clump Cultures: Its Structure and Role in Organogenic Potential of Single Leaf Cultures

Light microscopy demonstrated that the apparently amorphous,achlorophyllous tissue at the base of in vitro shoot clump cultureof Narcissus was comparable in structure to the basal plateof Narcissus bulbs. Both had very complex vascularisation andsmall, densely packed parenchymatous cells. In shoot clump cultures, primordia were produced by meristematiczones at the surface of this achlorophyllous tissue, very closeto the base of leaves. Single leaf units excised from the invitro shoot clump cultures with a wedge of basal achlorophylloustissue were highly organogenic when used as secondary explantsfor in vitro culture of Narcissus. No organogenesis occurredin the absence of the leaf base and achlorophyllous (basal plate)tissue and little organogenesis occurred unless the leaf baseand basal plate tissue were immersed in the culture medium (i.e.explants inoculated into liquid medium or upright in agar-solidifiedmedium). After two 5-week culture passages in liquid medium, more thanfive leaves were produced per leaf base inoculated. Thus rapidmicropropagation of Narcissus can be achieved using only thebase of single leaf units excised from shoot clump cultures.Copyright1993, 1999 Academic Press Anatomy, basal plate, bulb, in vitro, leaf culture, Narcissus, organogenesis     

Acclimation of Trees to Pollution Stress: Cellular Metal Tolerance Traits

Cell suspension cultures were established from shoot explantsof mature trees of Acer pseudoplatanus L. (sycamore) at a sitecontaminated by aerial deposition of copper and cadmium frommetal processing industry, and from the same species at uncontaminatedsites. The responses of cell cultures to elevated metal concentrationsin growth media differed markedly according to site of origin.Both Cu and Cd, applied singly at concentrations of 10–15mg l^–1, inhibited growth and were toxic to cultures originatingfrom the uncontaminated sites, but not to cultures from thecontaminated site. This metal tolerance trait in the culturesfrom the contaminated site was stable through repeated sub-culturing.It could also be induced in one culture originating from thereference uncontaminated site, by gradually exposing the cultureto increasing concentrations of Cu. A reduced level of metalremoval from the media was found in tolerant cultures, comparedto non-tolerant cultures. The results of these experiments demonstratethe occurrence of an alteration of gene expression in responseto pollution stress, suggesting that metal tolerance may beinduced within shoot meristems in vivo. It also represents thefirst example of non-mycorrhizal adaptation to metal toxicityidentified in woody plants. Trees, pollution, metal tolerance, acclimation, plant tissue culture, Acer pseudoplaianus L., sycamore     

Experimentally Synthesized Plant Chimeras I. In vitro Recovery of Nicotiana tabacum L. Chimeras from Mixed Callus Cultures

Experiments were conducted to develop techniques for synthesizingchimeras between plants of known genotype by utilizing in vitrotechniques Chimeral calli composed of green and albino tobaccocells were obtained by initiating callus tissue from mixturesof albino and green cotyledons, hypocotyls, callus culturesand cell suspensions The most effective mixing of genotypesoccurred when callus was derived from mixed filtered cell suspensionsUpon shoot regeneration, chimeral calli yielded 1317 non-chimeraland four chimeral plants Chimeras may have arisen as a resultof experimental procedures or possibly from spontaneous chromosomalabnormalities since leaves of some albino control plants occasionallyproduced small green islands of cells Explanations for the recoveryof a high percentage of non-chimeral shoots are presented Tobacco, callus cultures, cell suspensions, tissue culture, shoot apical meristems, somatic-crossing over     

Temporal and Spatial Heterogeneity in the Accumulation of Anthocyanins in Cell Cultures of Catharanthus roseus (L.) G.Don.

The accumulation of anthocyanins, a group of pigmented secondarymetabolites, in cell cultures of the Madagascar periwinkle Catharanthusroseus has been investigated. In these cultures it was foundthat anthocyanin accumulation was restricted to the post-divisionphase of the culture growth cycle, during which the culturesbecame deep purple in colour. As a result of anthocyanin visibilityit has been possible to ascertain that accumulation of thesemetabolites occurred in only a small proportion of the cellpopulation. Approximately 10% of cells regularly accumulateddetectable levels. Considerable variation within this ‘productive’population was observed and using a standard integrating microdensitometerit has been possible to quantify directly this heterogeneityand compare it with data obtained from whole plants. Analysishas revealed that the variation in both intracellular anthocyanincontent and concentration in cell cultures was much greaterthan that observed within tissues of mature plants. Significantdifferences in mean values were however found between the wholeplant tissues. The relevance of this temporal and spatial heterogeneityobserved in vitro to our understanding of the control of secondarymetabolite accumulation and to the potential use of tissue culturesystems as a means to produce these compounds is discussed. Key words: Heterogeneity, anthocyanins, cell culture     

Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601

Tomato explants were transformed with Agrobacter rhizogenesR1601 and root clones used to initiate longterm cultures inliquid and on solid media. The liquid cultures were maintainedfor 50 passages over 25 months in the presence and absence ofkanamycin. During this time the clones retained their high growthrates and antibiotic resistance phenotypes. The nptll gene wasdetected by PCR and dot blot hybridization in DNA from clonesat the 21st passage, and NPT-II enzyme activity was detectedin cell extracts prepared at the 45th passage. The solid cultureswere main tained for 12 passages over 12 months, either withcontinual selection, or with alternation during the last sixpassages between selective and non-selective media. The nptllgene was detected by PCR in DNA from cultures at the 5th passageand NPT-II enzyme activity was detected in cell extracts ofvigorously growing clones from the 12th passage. Passages inthe absence of selection had no discernible effect on the stabilityof the transformed state. The results indicate that the Ri-transformedstate can be maintained in tomato root clones during long periodsof culture. Key words: Agrobacterium rhizogenes, long-term cell culture, root clones, tomato, transformation     

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

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

Comparison of the flowering behavior of the long-day plant Lemna gibba G3 from different laboratories

In an effort to determine the cause for the wide discrepanciesin the level of flowering response reported for the long-dayplant Lemna gibba L., strain G3, cultures of L. gibba G3 wereobtained from the laboratories of W. S. Hillman (G3-H), R. Kandeler(G3-K), Y. Oota (G3-O) and and A. Pieterse (G3-P) and comparedto the L. gibba G3 (G3-C) from this laboratory. Under continuouslight all cultures gave FL% values of 77 or above, and on a9L:15D short-day treatment, all cultures were completely vegetative.However, on daylengths of 10 to 12 hr, small but statisticallysignificant differences were obtained for the different cultures.The critical daylength curves for G3-G, which showed the shortestcritical daylength, and G3-K, which showed the longest criticaldaylength, differed by approximately one hour. Salicylic acidtreatment caused flower promotion in each culture, but statisticallysignificant differences were obtained between some of the culturesin their response to salicylic acid. It is concluded that the large discrepancies in the floweringresponses of L. gibba G3 that have been reported are due primarilyto differences in culturing methods and counting proceduresin the different laboratories. However, the results also indicatethat there may be distinct cultures of L. gibba G3 that exhibitsmall physiological and/or genetic differences that would makeprecise quantitative comparison between different laboratoriesvery difficult. (Received January 23, 1979; )     

Electronic Measurement of Plant Cell Number and Size in Suspension Culture

The rapid and accurate determination of cell number and sizeis very desirable for obtaining data on growth kinetics andchanges in the physiological state of a culture. The CoulterCounter has been widely applied to bacterial and animal cellsuspension cultures to determine electronically cell numberand cell size. The application of such techniques to plant cellsuspension cultures is discussed in the following paper anddemonstrated for cultures of Pauls Scarlet Rose (Rosa sp.) andsoybean (Glycine max L.). A new formula for coincidence correctionsis suggested.     

Assimilation numbers in cultures of marine phytoplankton

During exponential growth in batch culture, assimilation numbersof eleven algal species ranged from 1.6–20.8, with a meanvalue of 5.3 g C/g Chlorophyll a/hr. The highest assimilationnumber of 20.8 g C/g Chlorophyll a/hr was observed in Coccolithuspelagicus, due to the relatively low concentration of chlorophylla/cell. The assimilation number declined from exponential tostationary phase in batch cultures for ten algal species, butincreased with age in batch culture in Amphiprora paludasa (abenthic diatom). The assimilation number declined with decreasinggrowth rate in nitrate-limited chemostat cultures of Phaeodactylumtricornutum and in iron-limited chemostat cultures of Phaeodactylumtricornutum and Isochrysis galbana.     

Histochemical Localization of Citral Accumulation in Lemongrass Leaves (Cymbopogon citratus(DC.) Stapf., Poaceae)

Lemongrasses (Cymbopogonspp., Poaceae) are a group of commerciallyimportant C₄tropical grasses. Their leaves contain up to 1.5%(d.wt) essential oils with a typical lemon-like aroma, consistingmainly of citral (a mixture of the isomeric acyclic monoterpenealdehydes geranial and neral). To specifically locate the sitesof citral accumulation in lemongrass we employed Schiff's reagent,which reacts with aldehydes and gives a purple-red colorationwith citral. Using this technique, single oil-accumulating cellswere detected in the adaxial side of leaf mesophyll, commonlyadjacent to non-photosynthetic tissue, and between vascularbundles. Cell walls of these oil cells are lignified. Our resultssuggest that citral accumulation takes place in individual oilcells within the leaf tissues.Copyright 1998 Annals of BotanyCompany Lemongrass;Cymbopogon citratus; Poaceae; oil cells; histochemistry; citral; aldehydes; Schiff's-reagent.     

The Role of Glucose on Flower Bud Formation in Thin-Layer Tissue 12Nicotiana tabacum L.

The effect of glucose on flower bud formation was studied inthin-layer tissue cultures of epidermal strips from flower stalksof Nicotiana tabacum L. cv. Samsun. A minimum concentration of 30 mol m^–3 glucose in the MS-mediumcontaining 1.0 mmol m^–3 of both NAA and BA was necessaryfor flower bud formation. With 150 mol m^–3 glucose a minimumstay of 10 d was required for optimal flower bud formation. Withholding glucose for a limited period at different time intervalsafter the onset of culture caused a delay in flower bud formationand did not affect previous development on glucose. The resultsindicated that competence for flower bud initiation is not restrictedto the early stage of culture. The process may start at anytime later at the appropriate glucose concentration. However,for both optimal initiation and further development of flowerbuds the presence of a metabolizable sugar is required. Incubationof the tissue on glucose is associated with higher respirationrate. Key words: Flower formation, Glucose, mannitol, Nicotiana tabacum, Respiration, tissue culture     

Melanin Biosynthesis Inhibitors from Tarragon Artemisia dracunculus

The EtOH extract of tarragon Artemisia dracunculus, a perennial herb in the family Asteraceae, was found to potently inhibit α-melanocyte-stimulating hormone (α-MSH) induced melanin production in B16 mouse melanoma cells. Bioassay-guided fractionation led to the isolation of two alkamide compounds, isobutyl (1) and piperidiyl (2) amides of undeca-2E,4E-dien-8,10-dynoic acid. The respective EC₅₀ values for melanin biosynthesis inhibition were 1.8 and 2.3 μg/mL for 1 and 2.     

Inheritance of Chloramphenicol Resistance, a Trait Selected in Cell Cultures of Nicotiana Sylvestris. Speg. and Comes

Three cell lines with improved resistance to growth inhibitionby chloramphenicol were selected from cell cultures of Nicotianasylvestris. Resistance was retained in callus cultures of twoout of three plants regenerated from one of the lines, but notin cultures of plants regenerated from the other two lines.Sexual progeny of the two resistant plants were either sensitiveor showed slow segregation for chloramphenicol resistance. Incallus from only two of the seedlings was inheritance of chloramphenicolresistance clearly demonstrated. Nicotiana sylvestris, cell culture, choramphenicol resistance