The Relationship between Growth Rate, Differentiation and Alkaloid Accumulation in Cell Cultures

The relationship between alkaloid content, growth rate and differentiationwas investigated in seven solanaceous species in culture. Evidencewas obtained for an inverse correlation between the growth phaseon the one hand and alkaloid content and cell organization (aggregation)on the other. Linear phase and stationary phase cells of Daturainnoxia Mill, incorporated DL-(1_–14C)ornithine-HCl differentiallyinto protein and alkaloids with proportionately more label foundin alkaloids in the stationary phase cells. On transferringcallus of D. innoxia from high auxin medium (containing 10–6M 2, 4–D) to low auxin medium (containing 10–6 M,2, 4–D), the alkaloid content dropped concomitant witha decrease in chlorophyll content and an increase in growthrate and friability. The nature of the interrelationships betweengrowth, differentiation and accumulation are considered. Key words: Tropane alkaloids, Accumulation, Growth, Differentiation     

Organogenesis, Differentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenicand organogenic calli induced with nine different cytokinin/auxincombinations. Alkaloid colour reagents localized tropane alkaloidsin the vascular regions which had large cells in the secondaryxylem of the basal stem sections of the non-rooted shoots. Tropanealkaloids were localized in shoots regenerated from calli inducedwith two different cytokinin/auxin combinations. No alkaloidswere localized in shoots regenerated from calli induced withother cytokinin/auxin combinations. However, only nicotine wasdetected by GC-MS in the non-rooted shoots regenerated fromcalli induced with three different cytokinin/auxin combinations.Tropane alkaloids were also localized in xylem cells of rootsregenerated from calli induced with two different cytokininand auxin combinations independently. The presence or absenceof nicotine, hyoscyamine and scopolamine in different culturedplant materials was confirmed by GC-MS, indicating that althoughthe root is the main site for alkaloid biosynthesis, with suitablecell differentiation, alkaloid biosynthesis may take place incultured shoots without root initiation. Copyright 2000 Annalsof Botany Company Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate     

Necessity of a Functional Octadecanoic Pathway for Indole Alkaloid Synthesis by Catharanthus roseus Cell Suspensions Cultured in an Auxin-Starved Medium

The effect of methyl jasmonate (mJA), jasmonic acid and traumaticacid, derivatives of the octadecanoic pathway, on the productionof alkaloids by cell suspension cultures of Catharanthus roseusL. (G) Don was investigated. Cells cultured in the presenceof auxin (m-cells) did not accumulate alkaloids. The additionof exogenous mJA to m-cells restored the ability to producealkaloids. In cells cultured in a 2,4-D-starved medium (p-cells),exogenous mJA greatly increased alkaloid production. Similardata were obtained for jasmonic acid. In contrast, traumaticacid had no effect on alkaloid production. The sensitivity ofcell suspension cultures to exogenous mJA was restricted tothe first four days of subculture corresponding to the activegrowth phase, whereas the alkaloid accumulation occurred onlyduring the stationary phase of the subculture (days 6 to 10).When p-cells were treated with octadecanoic pathway inhibitors,the ability to produce alkaloids was strongly reduced. The additionof exogenous mJA always restored the ability to produce alkaloids.These data suggest that in response to auxin depletion, endogenousmJA could be produced and act by linking physiological eventsthus leading to alkaloid biosynthesis activation. (Received May 31, 1997; Accepted December 7, 1997)     

Auxin Relations of the Woody Shoot: The Distribution of Diffusible Auxin in Shoots of Apple and Plum Rootstock Varieties

Surveys have been made of diffusible auxin in the stem tissuesof growing shoots of apple and plum rootstock varieties. Usingagar plates as carriers auxin was collected from the lower surfaceof isolated stem sections and assayed by the Avena curvaturemethod. The stool and layer shoots studied grew for severalmonths producing many leaves and reaching considerable lengths.The data provide information on selected internodes and showthe auxin status of the shoot at various times during growth,and the auxin gradients down the stem at these various times.Free auxin content of the shoot apex was consistently less thanthat of the internodes below. In 1946 auxin content declinedthroughout growth with a steady positioning of the auxin peakin the upper shoot. In 1947, following a period of drought,when growth almost ceased, a secondary auxin peak occurred positionedin lower internodes distant from the apex. This seasonal contrastwas reflected in the auxin relations of the individual internode,and was observed both in apple and plum. The nature of the auxindecline below the peak region, and the total disappearance offree auxin from the shoot as growth subsides, is discussed.The reappearance of free auxin in mature internodes, which hasnot been transmitted from the stem apex, implies either derivationfrom a stored state or the ability of the internode to produceits own auxin.     

The Control of Vascular Branching in Coleus 2. The Corner Traces

Corner trace connections are less well defined than those ofthe side bundle in Coleus, the locations of branch points, branchpartners, and number of connections made by a corner trace beingmore variable. The auxin balance between corner traces was alteredby leaf removal and by application of exogenous auxin. Branchingof new strands was shifted toward the pre-existing strand withthe lower auxin flux, but only within a narrow range of developmentalstages and with the imposition of a large auxin imbalance. Branchingoccurred only in nodal regions, as in control plants. Thus,auxin balance can be made to control xylem strand branching,but it does not account fully for the control of vascular branchingin intact plants. In the intact pattern, corner trace branchesappear to be directed toward the pre-existing strand with thehigher auxin flux. It is proposed that, in the vicinity of astrand with high flux, auxin is transported laterally withinthe nodal vascular cambium, facilitating vessel differentiationbetween strands in the derivatives of the vascular cambium.These vessels comprise the connections between traces. Coleus, vascular differentiation, vascular anatomy, vascular branching, vascular patterns, auxin, auxin balance, node     

Root-shoot Hormone Relations II. Changes in Endogenous Auxin Concentration produced by Flooding of the Root System in Helianthus annuus

Ethanol extracts of the shoot- and root-systems of Helianthusannuus were found to contain an auxin whose chromatographicbehaviour was similar to that of indole-3-acetic acid. The concentrationof this auxin in the shoots of plants with waterlogged rootsystems was found after 14 days to have risen to over threetimes the control value. There followed a drop in auxin contentto reach the control figure. This effect of flooding of theroot system upon shoot auxin content was correlated with characteristicmorphological changes. The effect of flooding on the auxin contentof the root system was to delay the fall off in auxin contentnoted in the root systems of control plants.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

Recent Advances in Indole Alkaloid Production by Catharanthus roseus (Periwinkle)

Considerable progress has been accomplished in cell culturetechniques for production of indole alkaloids fromCatharanthusroseus. This paper reviews the advances made in this area. Keyeffects of nutrients, environmental effects, stress-inducingcompounds and strain selection techniques on the productionof alkaloids are summarized. Cultivation methods such as suspensioncultures, immobilization, and a novel biofilm configurationare compared. Economic considerations in the large scale productionof secondary metabolites are covered. Indole alkaloids, cellculture techniques, Catharanthus roseus. Key words: Indole alkaloids, cell culture techniques, Catharanthus roseus     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots

In 1933 Thimann and Skoog demonstrated exogenous auxin repressionof lateral bud outgrowth in decapitated shoots ofVicia faba. This evidence has given strong support for a role of auxinin apical dominance. Most, but not all, investigators have confirmedThimann and Skoog's results. In the present study, auxin treatmentswere carried out on ten different species or plant types, manyof which were treated with auxin in different forms, media andunder different light conditions. The Thimann–Skoog experimentdid work for most species (i.e. exogenous auxin did repressbud outgrowth) including thedgt tomato mutant which is knownto be insensitive to auxin in certain responses. Toxic auxinsymptoms were observed in some but not all species. The Thimann–Skoogexperiment did not work for greenhouse-grownColeus or forArabidopsis. Light was shown to reduce apical dominance inColeus andIpomoeanil . apical dominance; lateral bud outgrowth; axillary bud; auxin; IAA; decapitation; Vicia faba ; Ipomoea nil ; Pisum sativum ; Phaseolus vulgaris ; Lycopersion exculentum ; dgt ; Coleus blumei ; Arabidopsis thaliana ; Helianthus annuus ; Thimann–Skoog     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

Changes in o-Diphenol Oxidase During Fibre Development in Cotton

Quantitative and electrophoretic changes in o-diphenol oxidase(o-diphenol: O₂ oxidoreductase, E.C. 1 10,3.1) were studiedduring the entire period of cotton (Gossypium arboreum L. cv.Sanjay) fibre development. A significant increase in o-diphenoloxidase activity was recorded during the fibre initiation phaseand it is suggested that a shift in redox balance towards oxidationmay play an important role in fibre initiation. Low o-diphenoloxidase activity during elongation and its high activity duringthe phase of secondary thickening, together with isoenzyme patterns,suggest an important role of this enzyme in cotton fibre development.The role of o-diphenol oxidase in relation to auxin turnoverand redox balance is discussed. Gossypium arboreum, cotton, fibre development, o-diphenol oxidase, redox balance, auxin turnover     

Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

Polar auxin transport inhibitors, including N-1-naphthylphthalamicacid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have variouseffects on physiological and developmental events, such as theelongation and tropism of roots and stems, in higher plants.We isolated NPA-resistant mutants of Arabidopsis thaliana, withmutations designated pir1 and pir2, that were also resistantto TIBA. The mutations specifically affected the root-elongationprocess, and they were shown ultimately to be allelic to aux1and ein2, respectively, which are known as mutations that affectresponses to phytohormones. The mechanism of action of auxintransport inhibitors was investigated with these mutants, inrelation to the effects of ethylene, auxin, and the polar transportof auxin. With respect to the inhibition of root elongationin A. thaliana, we demonstrated that (1) the background levelof ethylene intensifies the effects of auxin transport inhibitors,(2) auxin transport inhibitors might act also via an inhibitorypathway that does not involve ethylene, auxin, or the polartransport of auxin, (3) the hypothesis that the inhibitory effectof NPA on root elongation is due to high-level accumulationof auxin as a result of blockage of auxin transport is not applicableto A. thaliana, and (4) in contrast to NPA, TIBA itself hasa weak auxin-like inhibitory effect. (Received April 12, 1996; Accepted September 2, 1996)     

Purine Alkaloids of the Fruits of Camellia sinensis L. and Coffea arabica L. during Fruit Development

The amounts of two purine alkaloids, caffeine and theobromine,in the fruit of tea (Camellia sinensis L.) increased markedlyduring the growing season until the fruit was full-ripened anddried. In the dry fruit, the pericarp contained the most alkaloids,but there were also considerable amounts in the seed coat and,to a lesser extent, the fruit stalk and the seed. The shed seedsalso contained significant amounts of the alkaloids, especiallyin the seed coats. In contrast with the dry fruit of tea, seedsand pericarp of coffee (Coffea arabica L.) fruit contained aconsiderable amount of caffeine and a small amount of theobromide.A small amount of theophylline was also present in the pericarpof the ripened fruit. Relationships between growth and purinealkaloid content in tea and coffee fruits and their roles duringseed formation are discussed. Camellia sinensis L., tea, Coffea arabica L., coffee, purine alkaloids, fruit development, seed, seed coat, caffeine, theobromine, theophylline     

Modelling of quinolizidine alkaloid net flows in Lupinus albus and between L. albus and the parasite Cuscuta reflexa: new insights into the site of quinolizidine alkaloid synthesis

The present work reveals new and completely different conclusionsabout the alkaloid economy of symbiotically fed Lupinus albusand L. albus parasitized by Cuscuta reflexa in the study periodof 43–55 d after sowing of lupin. Net flows of alkaloidswithin lupin and between host and parasite were calculated usingthe molar ratio of alkaloid nitrogen: total nitrogen combinedwith known net flows of nitrogen in the transport fluids andanalysing alkaloid accumulation in plant organs by HRGC. Incontrast to previous studies, quinolizidine alkaloids were predictedto be synthesized mainly in the root of L. albus and to be predominantlytransported via xylem to the apical plant shoot organs. Parasitismby C. reflexa for 12 d induced a decline of alkaloid contentin the host L. albus up to 53% compared to control plants andalkaloid synthesis was halved—apparently due to a shortageof the precursor lysine. In spite of an additional decreasein nitrogen levels at the second harvest, the host-parasitesystem showed a1.3-fold higher alkaloid content than the controlplants, 63% of the total alkaloids being attracted by Cuscuta.This indicates (a) restriction of catabolic processes withininfected lupins, (b) a massive shift of nitrogen metabolismin the direction of alkaloids and (c) an enormous sink potentialof Cuscuta for nitrogenous compounds. Although xylem was foundto be the main translocation system for alkaloids, the modellingof alkaloid flows predicts Cuscuta to derive only 4.5% of itstotal alkaloid supply from the xylem and 95.5% from the phloem.By analogy with nitrogen flows, this finding requires xylemphloemtransfers which were assumed to occur within the stem axis oflupin. A similar proportion regarding the contribution of xylemand phloem to the supply of Cuscuta was obtained for the netflows of two selected alkaloids, lupanine and 13     

The Effect of Growth-regulating Substances and Light on Olive Callus Growth in vitro

In vitro olive callus cultures were established; auxin was notobligatory for the early development of the callus. However,after a number of subcultures, both an auxin and a cytokininwere needed for growth. IAA was the most efficient auxin ininducing growth, although 2,4-D was also very active. Very shortperiods of red-light illumination were sufficient to effectgreening of the tissue. A particularly high ratio of chlorophyllb/a was found under all light conditions tested. All light treatmentsstimulating chlorophyll synthesis caused a reduction in growthafter four subcultures.     

Characterization of plant growth substances in Citrus unshiu and their change in fruit development

The hormonal change in fruit development of Citrus unshiu wasinvestigated. A remarkable change in the content of auxin andABA was observed, suggesting that the hormonal balance may affectthe physiological abscission of the fruits. The occurrence ofIAA, IAM, IAA-Me and ABA in the young fruits was confirmed,while citrus auxin could not be detected. (Received August 14, 1975; )     

Variation of Alkaloid Content in Erythroxylum coca Leaves from Leaf Bud to Leaf Drop

A sequential study describing the content (%) of alkaloids inleaves of Erythroxylum coca var. coca Lam. from leaf bud developmentto leaf drop has not previously been conducted. The length oftime the leaf resides on the plant and its concurrent metabolicactivity also has not been defined. In the present study, cocaine,methyl ecgonine, hygrine, tropinone, trans -cinnamoylcocaine,cis-cinnamoylcocaine, tropacocaine and cuscohygrine were monitoredto determine: (1) their content and patterns of accumulationfrom leaf bud to leaf drop; (2) the time leaves resided on theplant; and (3) whether leaves were metabolically active untilleaf drop. E. coca plants were grown in a controlled environmentfor 37 months. Leaves similar in chronological age were extractedand analysed for alkaloid content by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS). Cocaine washighest in 7 d old rolled leaves (0·75%) and declinedto 0·39% at leaf drop. Cocaine, methyl ecgonine, hygrine,tropinone, trans -cinnamoylcocaine, cis-cinnamoylcocaine, cuscohygrineand tropacocaine content in 35 d old (mature) leaves was 0·61,0·59, 0·68, 0·08, 0·31, 0·55,0·52, and 0·05%. respectively. Cocaine, methylecgonine, hygrine, cis -cinnamoylcocaine, and cuscohygrine displayeda gradual decline from week 2 to week 36 of leaf duration. Tropinoneand tropacocaine were the least abundant of the alkaloids monitored.Cis-cinnamoylcocaine content exceeded cocaine at week 12, 16,and weeks 19 to 23 of leaf duration. Trans -cinnamoylcocainewas highest in rolled leaves (week 1) and in expanded leavesafter week 30. The monitored alkaloids appeared to be part ofthe metabolically active pool of the leaf. Leaves remained onthe E. coca plants for 36 weeks.Copyright 1994, 1999 AcademicPress Cocaine, methyl ecgonine, hygrine, tropinone, trans-cinnamoylcocaine, cis-cinnamoylcocaine, cusco-hygrine, tropacocaine, leaf bud, rolled leaves, expanded leaves, alkaloids, patterns, fluctuation, Erythroxylum coca var. coca, E, coca     

Analysis of the Potential Use of Androgenic Datura innoxia for the Development of Cell Cultures Producing High Amounts of Tropane Alkaloids

Normal and androgenic diploid Datura innoxia plants were selfedand the progeny was analysed for its leaf alkaloid content.Since the androgenic lines had originally produced very differentamounts of the tropane alkaloids, scopolamine and hyoscyamine,we were interested in determining whether this trait is transmittedby self-fertilization. The alkaloid content of the progeny wasfound to correlate well with that of the parental plants. Also,calli were initiated from leaf discs derived from plants withdifferent capacities for alkaloid biosynthesis. These were furthersubcultured for 2 years. Again, the same correlations in hyoscyamineand scopolamine content were observed. This indicates that itis possible to initiate callus with a high alkaloid contentstarting from actively alkaloid-producing androgenic Daturainnoxia plants. Key words: Datura innoxia, tropane alkaloids, androgenic plants, callus culture