Membrane Potential of Cultured Carrot Cells in Relation to the Synthesis of Anthocyanin and Embryogenesis

Membrane potentials of cultured carrot cells in culture mediumwere about –40 mV and did not change with addition ofsalts of addition (or depletion) of 2,4-dichlorophenoxyaceticacid (2,4-D). When the measurement was performed in the testmedium (containing low concentration of salts), the values werewidely distributed (from –60 to –110mV) and changedlargely with external concentration of K⁺ but not Mg²⁺ nor Ca²⁺.When the cells were fractionated by Ficoll density gradientcentrifugation, the membrane potential of the cells of higherdensity (> 14% Ficoll) was about –150 mV in the testmedium and did not change during embryogenesis with depletionof 2,4-D. The membrane potential of cells of lower density (bandingbetween 6– 10% Ficoll) was less negative (– 60 to– 110 mV) in the test medium. When these cells were transferredand cultured in medium containing zeatin but lacking 2,4-D,the membrane potential was shifted negatively by about 15 mVprior to anthocyanin synthesis. When 2,4-D was added to anthocyanin-synthesizingcells in the medium containing zeatin, a transient hyperpolarizationand subsequent depolarization of the membrane were observedprior to the inhibition of anthocyanin synthesis. (Received October 22, 1987; Accepted April 20, 1988)     

Effects of Monochromatic Radiations on Growth of Pelargonium Callus Tissue

Pith callus tissues were grown under continuous blue (450 mµ),green (545 mµ), red (650 mµ), and ‘white’(full-spectrum) light, and in the dark for 22 days at 27±2°C at energy levels of 15,000 ergs cm^–2 sec^–1. Mean increases in fresh weight of tissues grown under ‘white’and blue light were significantly greater than those of tissuesgrown in green and red light and in the dark. Tissues grownin the dark yielded mean fresh weight increases significantlylower than tissues grown under blue, red, and ‘white’light. No significant differences were shown between blue and‘white’, red and green, and green and dark treatmentsrespectively. Cell differentiation occurred in all treatmentsonly to the extent of vessel element formation. There were nodifferences in degree of differentiation between treatments. It was proposed that the high-energy reaction of photomorphogenesiswas in operation in the Pelargonium callus tissue. The resultsindicated the presence in the tissue of high-energy photoreceptor(s).The use of high-intensity, incandescent illumination for experimentalprocedures approximating natural conditions of irradiation wasindicated as desirable for pith callus tissues of Pelargoniumzonale var. Enchantress Fiat.     

Callus Induction and Plant Regeneration in Alstroemeria

Callus was induced from mature embryos of Alstroemeria cv. ‘Butterfly’cultured on MS medium supplemented with 2·0 or 4·0mg dm^–3 2,4–D or picloram and incubated at 25°Cin the dark. The effect of auxin concentration and precultureof embryos was studied. Callus was capable of regeneration aftertransfer to MS medium containing 4.0 mg dm°3 BAP. Shootsand whole plantlets were regenerated. The effect of growth regulators,used in the callus induction medium and the regeneration medium,on plant regeneration was studied Key words: Alstroemeria, callus, plant regeneration.     

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers.: 2. Nuclear Migration, Nucleolar Volume and Uptake of Tritiated Uracil

Sixty-eight per cent of nuclei in the cells of the upper fourlayers of carrot slices treated with heat-killed conidia ofBotrytis cinerea for 6 h followed by inoculation with live sporesfor 18 h, migrated to the cell face nearest to the treated surface,compared with 46 per cent in cells of control slices showinga wound-healing response only. Nucleolar volumes in the surfacecell layers of control slices increased from a mean of 1.0 µm³to 3.8 µm³ over 24 h, and in ‘induced’ slicesto 7.28 µm³. Using a 40 min pulse of [5–³H]uracil,there was an increase within 15 h of slicing in the number oflabelled nuclei in cells from control slices undergoing healing.Within 8 h after treatment of slice surfaces with heat-killedconidia, there was an accelerated incorporation of label into‘nuclear’ RNA. Slices from roots cold-stored for12 months failed to show an induction response and nucleolarvolumes did not increase more than in control slices. Theseresults are discussed in relation to active defence mechanismsin plant tissue. Botrytis cinerea, carrot, induced resistance, nuclear migration, nucleolar volume, RNA incorporation     

Changes in Activities of Enzymes Involved in General Phenylpropanoid Metabolism during the Induction and Reduction of Anthocyanin Synthesis in a Carrot Suspension Culture as Regulated by 2,4-D

The activities of enzymes involved in general phenylpropanoidmetabolism were followed in a carrot suspension culture duringthe induction and reduction of anthocyanin synthesis regulatedby 2,4-D. When no anthocyanin synthesis occurred in a mediumcontaining 2,4-D (+2,4-D medium), the activities of phenylalanineammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) increased1 day after transfer due to the transfer effect, but subsequentlydecreased and remained at a low level. Cinnamate-4-hydroxylase(C4H) activity showed a low level throughout culture. When cellswere transferred to a medium lacking 2,4-D (–2,4-D medium),the activities of PAL, C4H and 4CL increased and maximum activitiesof these enzymes were observed 6–7 days after transfer,when anthocyanin was most rapidly synthesized. When cells were cultured in the –2,4-D medium, the additionof 2,4-D immediately reduced the induced activity of PAL. PALactivity was super-induced by the transfer effect, while anthocyaninsynthesis decreased. The addition of intermediates of generalphenylpropanoid metabolism, with 2,4-D, to the medium 6 daysafter transfer to the –2,4-D medium did not promote anthocyaninsynthesis, whereas dihydroquercetin did promote it. Regulationof anthocyanin synthesis by 2,4-D is discussed in relation tochanges in enzyme activities involved in general phenylpropanoidmetabolism. ¹ Present address: Cell Science and Technology Division, FermentationResearch Institute, Agency of Industrial Science and Technology,Yatabe-machi, Ibaraki 305, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan.     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation     

Determinate Floral Buds of Plantain (Musa AAB) as a Site of Adventitious Shoot Formation

Floral buds of the ‘False Horn’ plantain clonesMusa (AAB) ‘Harton Verde’, ‘Harton Negra’,and ‘Currare’ terminate in a large single floralstructure. The apices of these floral buds are here designatedas determinate since they have lost the ability to produce additionalfloral initials or buds. Terminal peduncle segments can be culturedin a modified Murashige and Skoog (1962) medium supplementedwith N⁶-benzyl-aminopurine (5 mg I^–1). Under these conditions,this apparent inability to yield buds can be overcome as vegetativeshoot clusters form in the axils of the bracts. Rooted plantletsare obtainable by treating shoots with naphthaleneacetic acid(1 mg I^–1) and activated charcoal (0.025%). The adventitiousorigin of the shoots has been established. Musa cultivars, plantains, floral bud, adventitious buds, tissue culture     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Light-Induced Synthesis of Anthocyanin in Carrot Cells in Suspension: I. THE FACTORS AFFECTING ANTHOCYANIN PRODUCTION

Takeda, J. 1988. Light-induced synthesis of anthocyanin in carrotcells in suspension. I. The factors affecting anthocyanin production.—J.exp. Bot. 39: 1065–1077. A light-triggered anthocyanin-synthesizing system was establishedfor carrot cells in suspension. A few days after transfer ofthe cells to a 2,4-dichlorophenoxyacetic acid (2,4-D)-free mediumin the dark, light irradiation triggered anthocyanin synthesisand concomitantly stopped expansion growth. Over 90% of thecells synthesized anthocyanin without cell division. By loweringthe concentration of phosphate or both nitrogen and phosphateand delaying the time of onset of irradiation, the productionof anthocyanin per cell increased to a maximum level of 0–8µmol anthocyanin per 10⁶ cells. A change in the physiologicalstate of cells (light-insensitive to light-sensitive state)induced by the transfer to 2,4-D-free medium is suggested tobe a prerequisite for the light-triggered synthesis of anthocyanin. Key words: Anthocyanin production, cultured cells, Daucus carota, light-triggered, 2,4-D     

Effects of Inorganic Phosphate on the Utilization of Sucrose by Suspension-cultured Catharanthus roseus Cells

The metabolic fate of [U-¹⁴C]sucrose in suspension culturesof Catharanthus roseus cells was monitored for 96 h after thecells were transferred to fresh complete (‘+Pi’)or to phosphate-deficient Murashige and Skoog (‘–Pi’)medium. Sucrose was hydrolysed extracellularly to glucose andfructose. The rate of uptake of sugars by the cells was 1.5–3times higher in ‘+Pi’ culture than in ‘–Pi’culture. Little difference in the rate of incorporation of radioactivityinto the ethanol-soluble fraction was found between the ‘+Pi’and ‘– Pi’ cultures during the initial 24h of culture, but after 48 h the rate in ‘ +Pi’cultures was higher than that in ‘–Pi’ cultures.Incorporation of radioactivity into ethanol-insoluble macromoleculeswas always significantly higher in the cells in ‘+Pi’cultures than in those in ‘–Pi’ cultures.The results suggest that Pi strongly affects the utilizationof sugars by cultured plant cells through the stimulation oftransport of sugars as well as through the activation of metabolism. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, sucrose, transport, metabolism     

Behavioral responses of the brittle star Ophiura ophiura to amino acids, acetylcholine and related low-molecular-weight compounds

Feeding behavior of the brittle star Ophiura ophiura includesorienting posture, orienting movements, arm ‘walking’,changing the direction of ‘walking’ arm coilingand ingestion. All sequential behavior patterns were releasedor enhanced by single low-molecular-weight compounds. Stimuliwhich released ‘walking’ behavior at high concentrations(10^–4 M) in all the test animals are listed in decreasingorder of sensitivity: sarcosine, glycine, urea, L-valine, L-leucine,L-methionine, L-homocysteine, L-norvaline, L-norleucine, L-threonine,L-serine, S-methyl-L-cysteine, L-proline. Threshold values forsingle amino acids were as much as 100 times different in differentindividuals and ranged from 3 x 10^–9 to 3 x 10^–7M for the most effective stimulus, sarcosine, and from 10^–6to 10^–4 M for proline. Above 10^–5 M, only L-prolineregularly released a second behavior pattern, the arm coilingresponse, which temporarily inhibited the ‘walking’behavior. Behavioral thresholds for the ‘walking’behavior for L(+)-lactate and L-alanine were higher than thosefor the orienting movements. Thyoglycolic acid and ß-alaninereleased tube feet walking, which is not part of the feedingbehavior. Structure—activity comparisons were studied at estimated10^–5 M concentrations. Gycline, sarcosine, L-valine, L-norvaline,L-leucine, L-isoleucine, DL-norleucine and DL-homocysteine releasedarm ‘walking’ behavior in more than 75% of all thetests. With the exceptions of S-methyl-and S-ethyl-cysteine,and glycine methylester, derivatives of amino acids were noteffective behavioral stimuli in Ophiura ophiura. L-Isomers ofvaline and leucine regularly stimulated the ‘walking’behavior while their D-isomers were effective in some testsand ineffective in others. Acetylcholine iodide, acetyl-ß-methylcholine chloride and choline phosphate chloride regularly released‘walking’ behavior at concentrations above 10^–5M.     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. I. Influence of Pretreatment, Culture Medium and Culture Incubation Conditions on Callus Production and Differentiation

This study was conducted with Lolium temulentum, Festuca pratensis,and the two hybrids L. multiflorum x F. pratensis ‘Elmet’and L. perenne x F. pratensis ‘Prior’. In a comparisonof various durations (7–42 d) of pretreatment at 4 or7 °C the highest yield of microspore-derived callus of L.temulentum was obtained after pretreatment of spikes at 7 °Cfor 28 d, conditions which also proved optimal for panicle pretreatmentwith F. pratensis. For ‘Elmet’, durations of 21–42d were optimal, and for ‘Prior’ the responses tendedto decline with increasing duration. In L. temulentum addition of charcoal (1–2 g l^–1)to medium containing 2, 4-D and KN wa     

A Cytochrome P450 Mediated Naringenin 3'-Hydroxylase from Sweet Orange Cell Cultures

A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing themetabolism of naringenin to eriodictyol in Citrus sinensis (L.)Osbeck cv. ‘Hamlin’ cell suspension cultures wasshown to be a cytochrome P450 enzyme. This reaction requiredO₂ and NADPH and was inhibited by CO, with partial reversalof CO-inhibition by light at 450 nm. Cytochrome P450 contentranged from 10–20 pmol (mg microsomal protein)^–1.The F3'H reaction was shown to be linear in regard to proteinconcentration between 2.5 and 25 µg of microsomal protein.The optimum pH for the reaction was 7.4–7.6 and the temperatureoptimum was between 30 and 37°C. The apparent K_m and V_maxfor naringenin were 24 µM±3.2 and 81.4±7.9pmol eriodictyol min^–1 (mg protein)^–1, respectively.The microsomal F3'H was also capable of forming dihydroquercetinfrom dihydrokaempferol (40 pmol min^–1 (mg protein)^–1)and of quercetin from kaempferol (3.25 pmol min^–1 (mgprotein^–1). Cytochrome c and ketoconazole were the bestinhibitors of WH activity followed by piperonyl butoxide anda-naphthoflavone. Light was shown to be an inducer of the F3'Halmost doubling the specific activity and increasing the microsomalcytochrome P450 content by 30% over that of dark grown cells.F3'H activity was also confirmed in microsomal preparationsof young (new flush) leaves from ‘Hamlin’ treesand flavedo of ‘Hamlin’ oranges, ‘Marsh’grapefruit, and ‘Lisbon’ lemon. No activity wasobserved in older, hardened leaves and albedo of all the fruittested. Initiation of embryogenesis in the ‘Hamlin’cell suspension cultures by switching from a sucrose mediumto a glycerol-based medium resulted in the down-regulation ofF3'H. ¹Mention of a trademark, warranty, proprietary product, or vendordoes not constitute a guarantee by the U.S. Department of Agricultureand does not imply its approval to the exclusion of other productsor vendors that may also be suitable.     

Pattern of Respiration of a Perennial Ryegrass Crop in the Field

‘Dark’ respiratory losses of CO₂ were measured ona one year old sward of S24 perennial ryegrass (Lolium perenneL.) at intervals during a 74 day reproductive growth period,between April and June, and a 21 day vegetative growth period,in July and August. Part of the sward was shaded for one weekbefore measure ments commenced. Measurements of ‘dark’respiration continued for 46 hand it was possible to distinguishtwo components which are designated ‘maintenance’and ‘synthetic’ ‘Maintenance’ respiration was taken to be the meanrate of CO₂ efflux after 40–46 h darkness. When calculatedon a plant d. wt basis at 15°C it ranged between 6 to 32mgCO₂ g^-1 day^-1 during reproductive growth and 10–14 mgCO₂ g^-1 day^-1 during vegetative growth. During reproductivegrowth, sward protein content ranged between 7–23 percent and when maintenance respiration was recalculated on thebasis of protein content it changed relatively little throughoutthe growth period (90–140 mg CO₂ g pro tein^-1 day^-1);the value for vegetative growth ranged between 70–100mgCO₂ g protein-day^-1. Total ‘synthetic’ CO₂ flux was determined duringreproductive growth and a rate of ‘synthetic’ CO₂flux was determined during both reproductive and vegetativegrowth. Between 15 and 35 per cent of the CO₂ fixed in the previousphotoperiod was lost in ‘synthetic’ respirationof above-ground material in reproductive swards. Previous shadingincreased the proportion of ‘synthetic’ CO₂ lossfrom above ground. The rate of ‘synthetic’ CO₂ outputduring the first hours of darkness increased with amount ofCO₂ fixed in the previous photoperiod, although it was not proportionalto it. There is some evidence that assimilate is ‘carried-over’from one photoperiod to the next.     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

Relative Sensitivity and Tolerance of some Gladiolus Cultivars to Sulphur Dioxide

Five Gladiolus cultivars, namely ‘Aldebaran’, ‘BrightEye’, ‘Illusion’, ‘Manisha’ and‘Manmohan’, were exposed to 1 and 2 µg l^–1sulphur dioxide to test their relative-sensitivity toleranceto the pollutant Plants were fumigated experimentally for 2h daily Foliar injury symptoms were observed first in ‘Manisha’followed by ‘Aldebaran’ and ‘Illusion’at the higher dose Photosynthetic pigments and leaf extractpH were significantly decreased, particularly in ‘Manisha’and ‘Illusion’ Overall disturbances in the plantmetabolism due to SO₂ treatment led to retarded growth of plants,as evident from decreased shoot length and phytomass valuesThe order of sensitivity of the five Gladiolus cultivars toSO₂ was as follows, with the greatest first Manisha, Illusion,Aldebaran, Bright Eye, Manmohan Cultivars, Gladiolus, sensitivity, sulphur dioxide, tolerance     

ERRATA

Effects of coupled solute and water flow in plant roots withspecial reference to Brouwer's experiment. Edwin L. Fiscus. p. 71 Abstract: Line 3 delete ‘interval’ insert‘internal’. p. 73 Materials and Methods: line 6: delete ‘diversion’ insert ‘division’ line 9 equation should read J_v=L_p P–RT(C⁰–C¹). 74 Last line of figure legend: 10^–1 should read 10^–11. 75 Line 11: delete ‘seems’ insert ‘seem’. le 1 column heading—10⁶ should read 10¹¹. 77 delete ‘...membrane in series of...’ insert ‘membranein series or...’ Delete final paragraph.