The Accumulation of Volatile Oils in Whole Plants and Cell Cultures of Tarragon (Artemisia dracunculus)

Two important factors thought to be involved in determiningproduct yields in plant cell cultures are the genotype of theexplant material and the level of cell and/or tissue differentiationexhibited. Cell culture of A. dracunculus is reported for thefirst time and the accumulation of volatile oils in differentgenotypes and at various levels of differentiation are examined.The quantity and quality of oils accumulated varied markedlybetween plants of different types and with the level of anatomicaldifferentiation, both in planta and in vitro. The phenylpropene,allylanisole, the major component of commercial tarragon oil,is reported for the first time in disorganized cultures. Key words: Artemisia dracunculus, tarragon, tissue culture, essential oil, phenylpropenes, allylanisole     

Biochemical Basis of Different Sensitivity to Methotrexate in Daucus carota and Oryza sativa Cell Cultures

The biochemical basis of the different sensitivity to methotrexateof Daucus carota and Oryza sativa cell cultures has been investigated.Carrot cells have a dihydrofolate reductase activity about tentimes higher than rice cells. In addition, they show a loweruptake rate of the inhibitor. No relevant differences have beenfound in the K_m value for the dihydrofolate of the two enzymesand in the degree of inhibition of their activity by methotrexate. Key words: Dihydrofolate reductase, Methotrexate resistance, Plant cell suspension cultures, Oryza sativa, Daucus carota     

Effects of Methyl Jasmonate on Shikonin and Dihydroechinofuran Production in Lithospermum Cell Cultures

Methyl jasmonate, when administered to Lithospermum erythrorhizoncell suspension cultures, was found to induce the productionof shikonin derivatives (the red naph-thoquinone pigments ofthe root) and dihydroechinofuran (an abnormal metabolite ofgeranylhydroquinone). Culture experiments showed that methyljasmonate caused a rapid increase in the activities of enzymesinvolved in the biosynthesis of shikonin such as p-hydroxybenzoategeran-yltransferase, which was followed by the rapid accumulationof dihydroechinofuran and the delayed production of shikonin.The induction patterns observed were similar to those elicitedby oligogalacturonides in Lithospermum cells, suggesting thatjasmonic acid or its derivative may act as a signaling moleculein the elicitation of shikonin biosynthesis. Interestingly,however, the copper ion, which is essential for inducing shikoninbiosynthesis by oligogalacturonides, was not required for shikonininduction by methyl jasmonate ¹Present address: Laboratory of Molecular & Cellular Biology,Department of Agricultural Chemistry, Kyoto University, Kitashirakawa,Kyoto, 606-01 Japan     

Conversion of (+)-Dihydroquercetin to 3,4-cis-Leucocyanidin by a Reductase Extracted from Cell Suspension Cultures of Cryptomeria japonica

A soluble, NADPH-dependent reductase catalyzing the reductionof (+)-dihydroquercetin to 3,4-cis-leucocyanidin (5,7,3',4'-tetrahydroxyflavan-3,4-cis-diol)was demonstrated in an enzyme preparation from a cell suspensionculture of Japanese cedar (Cryptomeria japonica D. Don). TheK_m value for (+)-dihydroquercetin was 48µM. The enzyme,which was purified 26.2-fold, could also catalyze the reductionof (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The enzyme had a pH optimumof 7 and a molecular weight of 133,000. It was inhibited byCu²⁺ and iodoacetate, but not by p-chloromercuribenzoate. Duringthe growth stages of the cell suspension cultures, an increasein reductase activity proceeds an increase in procyanidin content,as might be expected. (Received November 25, 1987; Accepted April 11, 1988)     

Accumulation of Betacyanin in Phytolacca americana Cells and of Anthocyanin in Vitis sp. Cells in Relation to Cell Division in Suspension Cultures

In suspension cultures of Vitis sp., maximal accumulation ofanthocyanin was observed during the stationary phase. Accumulationof anthocyanin occurred in parallel with the cessation of celldivision under conditions such as a reduction of the concentrationof phosphate in the medium, or the presence of aphidicolin,an inhibitor of DNA synthesis. By contrast, in suspension culturesof Phytolacca americana, aphidicolin inhibited the accumulationof betacyanin and cell division. When aphidicolin was removedfrom cells by washing, partially synchronized division of cellswas induced and the accumulation of betacyanin also occurred,in conjunction with cell division. In the absence of phosphatefrom the medium, cell division did not occur and accumulationof betacyanin also ceased. Readdition of phosphate to cellsstarved for phosphate induced both cell division and the accumulationof betacyanin. These results indicate a positive correlationbetween the accumulation of betacyanin and cell division inPhytolacca which contrasts with a negative correlation betweenthe accumulation of anthocyanin and cell division in Vitis. (Received April 17, 1989; Accepted December 23, 1989)     

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     

Fine Structure of Established Callus Cultures of Taraxacum officinale Weber

The nodular, brown callus cultures of Taraxacum officinale growslowly on a modified White's medium. A section of nodule revealsa meristematic layer bounded both internally and externallyby parenchymatous tissue. No other types of tissue occur withinthe callus. The cells of the inner parenchyma are often compressedand senescing, whereas in the outer tissue localized de-differentiationapparently contributes to the development of new nodules duringcallus growth. Fine-structural observations of both meristematic and parenchymatoustissues show the normal complement of higher plant cell organellesexcept for the apparent absence of cytoplasmic microtubules.The rough endoplasmic reticulum cisternae are often alignedparallel to the cell wall or in whorls and may show pores, thusresembling annulate lamellae. Numerous lipid bodies, up to 7µm wide, occur and these are sometimes invested by arraysof apparently membranous material. The mitochondria are frequentlyhighly branched and often show a scalariform arrangement ofcristae. The plastids show few internal membranes despite cultureof the callus under continuous illumination. Lomasomes are very common in all cells and in the parenchymatissue membranous wall bodies also occur. The latter bodiesare much larger than lomasomes and consist of wall overgrowthsin which vesicular, myelin-like or isolated membranous elementsare enmeshed in fibrillar material. It is suggested that membranouswall bodies may originate from the amalgamation and subsequentproliferation of several adjacent lomasomes. Taraxacum officinale Weber, dandelion callus cultures, fine structure     

Intercellular and Intercultural Heterogeneity in Secondary Metabolite Accumulation in Cultures of Catharanthus roseus following Cell Line Selection

Hall, R. D. and Yeoman, M. M. 1987. Intercellular and interculturalheterogeneity in secondary metabolite accumulation in culturesof Catharanthus roseus following cell line selection.—J.exp. Bot. 38: 1391–1398. Anthocyanin accumulation in a stock culture of Catharanthusroseus was consistently found, using microscopic and microdensitometrictechniques, to involve only c. 10% of the cell population. However,an analysis of 26 cell lines isolated from this culture hasindicated that all of the cells within the culture were, theoretically,capable of anthocyanin synthesis. Nevertheless, these linesdid display substantially different capacities for anthocyaninaccumulation. Detailed studies on individual cells from thesecultures have revealed that the variation in accumulation wasprimarily due to differing proportions of pigmented (i.e. productive)cells rather than differing mean intracellular anthocyanin concentrationswithin these cells. Both the proportion of productive cellsand the overall culture yield of the cell lines varied by >30-fold whereas the mean intracellular anthocyanin concentrationvaried by < 2-fold. The relevance of these results to thepossible control mechanisms involved in secondary metaboliteproduction in this and other culture systems is discussed. Key words: Catharanthus roseus, cell culture, anthocyanin, heterogeneity     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture     

Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Multiple Metal Resistance and Co-resistance in Acer pseudoplatanus L. (Sycamore) Callus Cultures

In vitro growth of Acer pseudoplatanus L. (sycamore) callustissue derived from shoot-tip explants and screened on metal-enrichedmedia was studied in an attempt to identify resistance traitswhich may explain the survival of trees at metal-contaminatedsites. Copper and Cd-resistance traits were identified in celllines originating from trees at a site with a relatively recenthistory of severe contamination by these metals, and Cd- andZn-resistance were identified in cell lines originating frommature trees at a mining spoil site with a much longer historyof exposure to elevated concentrations of these metals. In Zn-resistantcell lines, co-resistance to Ni was also found, even thoughthis metal was not elevated at the study site. This is the firstreport of multiple resistance and co-resistance to metals occurringat the cellular level in trees. The mechanisms of the measuredresistance traits remain unclear, although there was evidenceof reduced Cu and Ni uptake by resistant cell lines. It is concludedthat facultative adaptations allowing acclimation to metal stressmay be particularly significant for survival of mature trees;induction of metal resistance probably occurs in vivo in treesat metal-contaminated sites.Copyright 1995, 1999 Academic Press Acer pseudoplatanus, sycamore, callus, heavy metals, metal resistance, trees     

The Influence of Factors of the Aeration Complex and Light upon Fruit-body Form in Pure Cultures of an Agaric and a Polypore

Methods involving controlled temperature and illumination, continuouslyrenewed sterile culture atmosphere of defined gaseous compositionand vapour pressure, and automatic replacement of water lossfrom the medium have been applied to the analysis of sporophoreform in Collybia velutipes and Polyporus brumalis. In P. brumalisprogressive suppression of the pileus but enhanced stipe elongationoccurs as light intensity (over a certain range) or transpirationalwater-loss are reduced. These factors act additively. Cap expansionin C. velutipes requires light and is, in addition, inhibitedby an atmospheric staling agent removable by KOH and replaceableby CO₂. Data and observations on conditions affecting timesof production, numbers and dimensions of fruit-bodies are given,especially for P. brumalis. Other aeration factors are consideredbut shown to have no morphological effect.     

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     

In Vitro Selection for Salt Tolerance in Brassica juncea L. Using Cotyledon Explants, Callus and Cell Suspension Cultures

Salt-tolerant Brassica juncea L. cell lines or plants have beenselected by screening callus pieces, cell suspension culturesand cotyledon explants in vitro on high concentrations of NaCl.Callus-based selection was unsatisfactory, as only two out ofseven isolated clones retained tolerance after 3 months of subcultureon NaCl-free medium. Selections made via plated cell suspensionswere found to be more stable for salt-tolerance. AH selectedtolerant cell lines, however, failed to regenerate plantlets.A third selection method, employing cotyledon explants was basedon their high potential for regenerating multiple shoots. Outof a total of 2620 explants cultured on high salt media, threesurvived, showed sustained callus proliferation and each regeneratedone shoot. The salt-selected shoots withstood the stabilitytest after 3 months of growth and axillary bud multiplicationon NaCl-free medium. While one of these somaclones was morphologicallyabnormal and sterile, the other two could be reared to maturitywith normal seed set. Brassica juncea, tissue culture, in vitro selection, salt-tolerance, plant regeneration     

Growth Inhibition of Intact Plants and In Vitro Cultures of Tobacco by Culture Filtrates of Fusarium oxysporum f.sp. nicotianae

The culture filtrate of Fusarium oxysporum was able to induceseveral symptoms on tobacco plants that appeared during theactual pathological condition. Positive correlations were establishedfor the degree of culture-filtrate-induced symptoms developedin a range of tobacco cultivars and those caused by the livepathogen after inoculation into the same cultivars. Effectsof culture filtrate on the growth of intact plants, anthers,leaf discs and cell suspensions were examined to assist in understandingthe cellular basis of pathogenicity. The culture filtrate above25 % (v/v) was found inhibitory to the growth of leaf-disesand plated cell suspensions. No growth occurred above the 50%(v/v) level of the filtrate. Similarly, wilt symptoms were observedon the whole plants when the culture filtrate exceeded 25% (v/v).In vitro androgenesis was inhibited at a much lower concentration(12.5% v/v) of culture filtrate. Fusarium oxysporum f.sp, nicotianae, Nicotiana tabacum, fungal culture filtrate, will disease     

Investigations of Laticifer Differentiation in Tissue Cultures Derived from Asclepias syriaca L.

Callus cultures of Asclepias syriaca were established from stemexplants and grown in tissue culture. The culture medium onwhich the callus was grown was modified to produce either planfletsof superficial origin on the callus or embryoids which wereanalyzed to determine whether laticifers differentiated in thesestructures. Mature zygotic embryos and adult plants of A. syriacanormally possess a well-developed network of intrusively-growingnon-articulated branched laticifers that arise only once duringplant develop ment from initials differentiated in the youngheart stage embryo. Embryoids were derived from two differentculture media. These embryoids were observed to lack laticifers,although they were similar in their morphology in other respectsto zygotic embryos. Plantlets of superficial origin were formedon each of the media employed in this study. These plantletswere observed to possess laticifers that resemble those in normalshoots. Embryoids and induced shoots represent experimentalsystems in which it may be possible to control for the firsttime the differentiation of the laticifer as a cell type instructures similar to those present in the normal plant.     

Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

Plantlet Regeneration from Suspension and Protoplast Cultures of the Tropical Pasture Legume Stylosanthes guyanensis (Aubl.) Sw

Hypocotyl- and leaf-derived suspension cultures of Stylosanthesguyanensis were established and plants were regenerated fromthem, even after several sub-cultures. Protoplasts, enzymaticallyisolated from cell aggregates of a highly morphogenetic suspensionculture, synthesized new cell walls, divided and developed intocalli. Plantlets were regenerated from these calli after successivetransfers to solid maintenance and shoot induction media. Stylosanthes guyanensis (Aubl.) Sw, suspension culture, protoplasts, shoot regeneration, plantlet regeneration     

Fractionation and Partial Characterization of Pectic Polysaccharides in Cell Walls from Liverwort (Marchantia polymorph) Cell Cultures

Konno, H., Yamasalu, Y. and Katoh, K. 1987. Fractionation andpartial characterization of pectic polysaccharides in cell wallsfrom liverwort (Marchantia polymorpha) cell cultures.—Jexp. Bot. 38: 711–722. Pectic polysaccharides were extracted from the starch-free cellwall preparation of cell suspension cultures of Marchantia polymorpha.The polysaccharides were fractionated by DEAE-Sephadex A-50ion-exchange chromatography yielding the five fractions, andthe degree of polymerization and glycosyl composition determinedfor each fraction. The neutral rich and acidic pectic polymerswere depolymerized by purified endoglucanase (l,4-ß-D-glucan4-glucanohydrolase, E.C. 3.2.1.4 [EC] .) and endopolygalacturonase(poly-l,4--Dgalacturonide glycanohydrolase, E.C. 3.2.1.15 [EC] ),respectively. The degraded pectic fractions were fractionatedby gel filtration chromatography on Bio-Gel A-5m and Bio-GelP-2, and glycosyl composition determined for each fraction.The results indicate that pectic polysaccharides contain glucose-richpolymer, rhamnogalacturonan and homogalacturonan in a ratioof 1:4:0–6. In addition, pectic polysaccharides were releasedas five pectic fragments from the cell walls by purified endopectatelyase (poly-l,4--D-galacturonide lyase, E.C. 4.2.2.2 [EC] ). Basedon the analysis of glycosyl composition of each fragment, thepectic polysaccharides of Marchantia cell walls are characterized Key words: Cell suspension culture, cell wall, liverwort, Marchantia polymorpha, pectic polysaccharides     

Salt Tolerance in Cell Suspension Cultures of the Halophyte Kosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures ofKosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonoushalophyte that grows in tidal marshes of the eastern UnitedStates. Growth of salinized cultures was significantly inhibitedat high (255 mol m^–3 NaCl), but not at lower externalsalinities. Adjustment of cell suspensions to Nacl was rapid,with the duration of the normal growth cycle unaffected by salinity.Maximum biomass was attained when cultures were exposed to NaClduring early log growth. Patterns of inorganic ion accumulationreflected the utilization of both Na⁺ and K⁺ as osmotica, withNa⁺ content substantially increasing when cells were grown atan external salinity sufficient to reduce growth. K⁺ uptakeselectivity was high and Na⁺/K⁺ ratios were low in salt-treatedcultures even though K⁺ content was somewhat lower comparedto unsalinized cultures. Free proline and microsomal lipid contentincreased in salt-treated cell cultures. Key words: Kosteletzkya virginica, halophyte, salt tolerance, cell suspension culture