Floral Induction in a Photoperiodically Neutral Duckweed, Lemna paucicostata Strain LP6: Role of Chelating Agents and Iron

Lemna paucicostata, strain LP₆, does not ordinarily flower underany photoperiodic schedule, when grown in the modified Bonner-Devirianmedium supplemented with 10^–4 M EDTA and 1% sucrose (thisis a medium which is otherwise satisfactory for short day inductionof flowering of other strains of this duckweed). However, when the ferric citrate concentration in the culturemedium containing EDTA was raised 10-fold over that in the normalmedium, a low but significant flowering could be initiated inthis duckweed, irrespective of the length of the photoperiod.A similar flowering response was obtained when ferric citratewas replaced by ferrous sulphate or ferric chloride, therebyindicating that the inductive effect of higher level of ferriccitrate on flowering in strain LP₆ is due to its iron component. Some flowering in this strain could be induced even in mediumcontaining normal level of iron, provided the level of EDTA,itself, was raised to 5?10^–4 (5% flowering) or to 10^–3M (12% flowering), but replacement of EDTA by EDDHA led to trulyremarkable effects. With EDDHA, flowering could be induced at very high levels (90%)under all photoperiodic regimes tried. Floral initiation couldbe obtained even with 10^–6 M EDDHA, though the optimallevel ranged from 5?10^–6 to 10^–5 M. (Received October 4, 1985; Accepted June 26, 1986)     

Effect of Spermidine and Methylglyoxal-bis (Guanyl-hydrazone) (MGBG) on In Vitro Pollen Germination and Tube Growth in Catharanthus roseus

Incorporation of polyamine-spermidine into the nutrient mediumat 10^–6 and 10^–5 M concentrations stimulates pollen-tubegrowth in vitro in Catharanthus roseus L. G. Don. MGBG, an inhibitorof spermidine biosynthesis, at 0.5 x 10^–3 and 1 x 10^–3M concentrations reduced the percentage of germination as wellas tube growth and at a concentration of 1.5 x 10^–3 Mgermination was totally inhibited. Pollen grains incubated inthe medium containing 1.5 x 10^–3 M MGBG, when transferredto a fresh medium with 10^–5 M spermidine, resulted in80% germination recovery, along with considerable tube growth.Experiments with actinomycin-D indicate that stimulation ofpollen-tube growth by spermidine may involve de novo synthesisof protein. Catharanthus roseus, pollen germination, tube growth, spermidine, MGBG, inhibition, actinomycin-D     

Direct Somatic Embryogenesis in Roots of Cichorium: Is Callose an Early Marker?

Direct somatic embryogenesis can be obtained from epidermaland cortical cells in roots from in vitro Cichorium plantlets.The first embryogenic cells are seen after six days of culturein darkness, at 35 °C, in a liquid medium supplemented withNAA (1 x 10^–7 M), 6-dimethylallyl-amino-purine (2·5x 10^–6 M), sucrose (0.03 M) and glutamine (1·7x 10^–3 M). Embryogenic cells undergo first a linear andthen a globular segmentation, with increasing cytoplasmic density.These cells and young embryoids show aniline blue fluorescence.SEM allows the same microglobular pattern to be seen on thesurface of young embryoids and on young microspores of Cichoriumused as controls. In this root system, callose deposition seemsto be an early marker in somatic embryogenesis. Somatic embryogenesis, callose, Cichorium     

Induction of Flowering in a Duckweed, Wolffia microscopica, under Non-inductive Long Days, by 8-Hydroxyquinoline

Wolffia microscopica, a duckweed, flowers in response to a singlephotoinductive SD cycle of 16 h dark and 8 h light. Floweringin W. microscopica could be induced, under non-inductive longdays, by 8-hydroxyquinoline (8-HQ). Flowering was initiatedwith 10^–6 M 8-HQ and maximum flowering (ca. 75%) was obtainedat 5 x 10^–6M level. Flowering was accentuated furtherwhen plants, supplied with 8-HQ, were subjected to SD cycles. (Received September 13, 1985; Accepted December 4, 1985)     

Behavioral determinations of thresholds for n-butyl acetate and nbutyl alcohol in the tiger salamander (Ambystoma tigrinum)

Two groups of tiger salamanders (Ambystoma tigrinum) were conditionedto respond to odorant-air mixtures of n-butyl acetate (8.9 x10^–5M) or n-butyl alcohol (6.7 x l0^–5M). They werethen given tests with various concentrations of the trainingodorants presented using a temporal forced-choice method ofascending limits. Results showed that reliable responses toodorant-air presentations were obtained with concentrationsof n-butyl acetate above 2.4 x l0^–7M and with concentrationsof n-butyl alcohol above 8.5 x 10^–8M. These results arein substantial agreement with previous dectrophysiological findings.     

Bl?tenbildung bei Lemna paucicostata 6746 durch kombinierte Anwendung von Abscisins?ure und GCG

To a certain extent the flowering of Lemna paucicostata 6746is evoked in continuous light by application of abscisic acid(ABA) and CCC. Moreover, the action of the combined substancesappears in two separate concentration ranges. In the range ofABA 2?10^–9 M/CCC 10^–7–10^–6 M floweringis initiated without inhibition of vegetative growth and proceedsonly in the presence of high intensity light and sucrose. Acombination of ABA 2?10^–5 M/CCC 10^–3M simultaneouslycauses a strong inhibition of frond multiplication. Here theeffect can be observed also under low intensity light conditionsand without sucrose in the medium. A range with flower inhibitingactivity lies between the two flower promoting concentrationranges. (Received November 16, 1972; )     

Release of Guttation Fluid from Passive Hydathodes of Intact Barley Plants. II. The Effects of Abscisic Acid and Cytokinins

Guttation was used as a non-destructive way to study the flowof water and mineral ions from the roots and compared with parallelmeasurements of root exudation. Guttation of the leaves of barley seedlings depends on age andon the culture solution. Best rates of guttation were obtainedwith the primary leaves of 6- to 7-day-old seedlings grown onfull mineral nutrient solution. The growing leaf tissue becomessaturated with K⁺ below 1.5 mM K⁺ in the medium, whereas K⁺concentration in the guttated fluid still increases furtheras K⁺ concentration in the medium is raised. At 3 mM K⁺ averagevalues of guttation were 1.4–2.4 mm³ h^–1 per plantwith a K⁺ concentration of 10–20 mM; for exuding plantsthe flow was 4.2–7.6 mm³ h^–1 per plant and K⁺ concentration35–55 mM. Abscisic acid (ABA) at 10^–6 to 10^–4 M 0–2h after addition to the root medium increased volume flow ofguttation and exudation and the amount of K⁺ exported. Threeh after addition of ABA both volume and amount of K⁺ were reduced.There was an ABA-dependent increase in water permeability (Lp)of exuding roots shortly after ABA addition. Later Lp was decreasedby 35 per cent and salt export by 60 per cent suggesting aneffect of ABA on salt transport to the xylem apart from itseffect on Lp. Benzyladenine (5 x 10^–8 to 10^–5 M)and kinetin (5 x 10^–6 M) progressively reduced volumeflow and K⁺ export in guttation and exudation and reduced Lp. Guttation showed a qualitatively similar response to phytohormonesas found here and elsewhere using exuding roots. Hordeum vulgare L., barley, guttation, abscisic acid, cytokinins, benzyl adenine, kinetin     

The Genetic Basis of Preference for Sweet Substances Among Inbred Strains of Mice: Preference Ratio Phenotypes and the Alleles of the Sac and dpa Loci

Five inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BI/6J andDBA/2J) were examined with two-bottle (48 h) preference ratiotesting across concentrations of sodium saccharin (3 x 10^–4M, 10^–3 M, 3 x 10^–3 M and 10^–2 M), d-phenylalanine(10^–3 M, 10^–2 M and 10^–1 M), and l-glutamine(10^–2 M, 3 x 10^–2 M, 10^–1 M and 3 x 10^–1M). Three consistent groupings of strains were observed acrosssubstances and concentrations:

1. C57BI/6J (preference at low andhigh concentrations);
2. BALB/cByJ and C3HeB/FeJ (preferenceat high concentrations);
3. 129/J and DBA/2J (preference at highconcentration for sodiumsaccharin and indifference to d-phenylalanineand l-glutamine).

If a single locus (presumably dpa or Sac) determines these phenotypes,there are likely to be three alleles. If two independent loci(presumably dpa and Sac) determine these phenotypes, an allelicassignment of Sac^b/dpa^+s for the C57BI/6J strain, Sac^b/dpa^–sfor the BALB/cByJ and C3HeB/FeJ strains, and either Sac^d/dpa^+sor Sac^d/dpa^–s for the 129/J and DBA/2J strains is suggested.Chem. Senses 20: 291–298, 1995.     

The NADP$-specific isocitrate dehydrogenase of Rhodospirillum rubrum

The NADP^$-specific isocitrate dehydrogenase was partially purifiedfrom photosynthetically-grown Rhodospirillum rubrum. The pHoptimum is between 7.5 and 9.0 in phosphate buffer. The apparentKm is 3.1x10^–5 M for isocitrate, 5.1x10^–5 M forNADP^$, 1.7x10^–5 M for manganese, 1.5x10^–4 M formagnesium, and 3.5x10^–3 M for inorganic orthophosphate.Arsenate exerts a slight inhibition. The Q₁₀ between 17.5°Cand 40°C is 1.62, and the energy of activation at 25°Cis 9.74 Kcal/mole. Glyoxylate and oxalacetate cause concertedinhibition of the enzyme activity. Various nucleotides inhibitthe activity. The kinetics of inhibition by ATP was found tobe mixed type with respect to NADP^$ and isocitrate, the K_i valuesbeing 1.17x10^–3 M and 1.10x10^–3 M respectively.The inhibition between ATP and orthophosphate is competitivewith a K_i of 10^–4M. Thiol binding reagents are inhibitory;this inhibition is reversed by cysteine or reduced glutathione. (Received October 1, 1971; )     

Regulation of Free Calcium Concentration in the Pitchers of the Carnivorous Plant Sarracenia purpurea: A Model for Calcium in the Higher Plant Apoplast?

The pitcher of the carnivorous plant Sarracenia purpurea L.contains an entrapped body of liquid within which its prey isdigested. Free calcium in the pitcher is derived from eitherthe pitcher walls or from prey falling into the pitcher; inthe absence of exogenous (prey-derived) calcium it will dependon the active and passive calcium regulatory properties of thepitcher walls and may to some extent therefore mimic calciumin the apoplast of plant cells. Using a calcium-specific electrode,the free calcium concentration of the pitchers of Sarraceniaplants was investigated and the effect of adding a variety ofconcentrations of calcium in water determined. The mean pitcherfree calcium concentration in vivo was 2.3 x 10^–5 M±2.5x 10^–5 M; when pitchers were washed and filled with watercontaining lower calcium concentrations, the concentration inthe pitcher water rose to 1–5 x 10^–5 M. When highercalcium concentrations (up to 1 x 10^–4 M) were added,the pitcher calcium concentration declined to 1–7 x 10^–5M. Concentrations of calcium above 1 x 10^–4 M were alsoreduced, but to a lesser extent. Metabolic inhibition of activeion transport, while inhibiting pitcher acidification, did notinhibit regulation of pitcher free calcium, suggested that itoccurs as a result of calcium exchange sites in the pitcherwalls. The data are discussed in relation to the physiologyof Sarracenia pitchers and to the usefulness of the pitcheras a model for free calcium in the higher plant apoplast. Sarracenia purpurea L., carnivorous plant, pitcher, free calcium, plant apoplast     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

The possible role of cyclic AMP in the control of genetic tumor induction

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolensx N. langsdorffii, grown aseptically on nutrient medium, weretreated with 1x10^–2 M cyclic adenosine 3': 5'-monophosphate(cyclic AMP). The incidence of tumor formation was scored atregular intervals subsequent to exposure. Cyclic AMP causeda significant reduction in the rate of tumor formation. In addition,untreated plants grown on nutrient medium were harvested atregular intervals after the seeds had been sown. Cyclic AMPwas extracted, partially purified, and assayed radioimmunologically.The endogenous level of cyclic AMP in stem tissue was highestin young seedlings, rapidly fell reaching a low point in 18day-old plants, and eventually leveled off. The presence ofindoleacetic acid (IAA) in the growth medium at a final concentrationof 2x10^–5 M prevented the decline in cyclic AMP that occurredin seedlings grown on unsupplemented medium. (Received May 21, 1976; )     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

Resemblance of growth substances to metal chelators with respect to their actions on duckweed growth

Action patterns of IAA, KIN and GA on the growth of Lemna gibbaG3 are similar to those reported for agents chelating both cupricand ferric ions. Relatively high doses of growth substances,e.g.10^–6 M IAA or KIN and ^–4 M GA, inhibit developmentof photoperiodically induced flower buds and antagonisticallypromote frond multiplication; whereas, at relatively low doses,e.g. 10^–9 M IAA or KIN and ^–5 M GA, they acceleratethe process occurring in the latter half of the induction periodto enhance flower induction. Complex forming abilities of IAA,KIN and GA with cupric and ferric ions are demonstrated spectrophotometrically.Moreover, the ferrous ion-dependent oscillatory change in reproductiveand vegetative photophilies of duckweed is eliminated by KINbut not by IAA and GA. Of the three growth substances tried,KIN alone shows an affinity for ferrous ions. (Received April 5, 1971; )     

Vessel element formation in cultured carrot-root phloem slices

The effects of light, auxin and cytokinin on vessel elementformation in phloem slices of carrot root were examined. When slices of carrot cultivars, ‘Nakamura-senko-futo’and ‘Yamada-hyakunichisenk6- naga’, preculturedin the dark on modified Murashige and Skoog's medium for twodays were cultured on a medium containing 5x10^–6 M 2,4-Din the dark, no vessel element formation occurred. When preculturedslices were cultured in the light with 5x10^–6M 2,4-D,vessel element formation was remarkable. But when 5x10^–7Mkinetin, benzyladenine or zeatin was added, vessel elementswere readily formed even in the dark. When slices were cultured in the light, a cytokinin-like substance(s)that causes vessel element formation was produced in the slices,then was released to the medium. The substance(s) was fairlystable to heat. In slices of carrot cultivars, kuroda-gosun, ‘Kintoki’and ‘Kokubu-senk6-6naga’, a different result forvessel element formation was obtained. When slices of thesecultivars were cultured on a medium containing 5x10^–6M2,4-D in the dark, vessel element formation was remarkable.It seemed, therefore, that these cultivars contain enough ofa cytokinin-like substance(s) to form vessel elements. In fact,vessel element forming activity was found in the alcohol extractof carrot root phloem from these cultivars. (Received June 8, 1971; )     

L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)     

Storage Organ Development in Radish (Raphanus sativus L.). 2. Effects of Growth Promoters on Cambial Activity in Cultured Roots, Decapitated Seedlings and Intact Plants

The radish varieties Cherry Belle and Long White Icicle wereused to investigate the role of the shoot and the effects ofsynthetic growth promoters in controlling cambial activity inthe seedling axis. Development was compared in excised roots, roots with hypocotylsattached and intact seedlings cultured aseptically on a nutrientmedium. No cambial divisions were seen in isolated radicleswhich had been cultured for ten days following excision butretention of hypocotyl tissue or the entire shoot resulted incambial activity and the production of secondary vascular tissues.Enriching the culture medium by raising the sucrose conantrationto 8% and including 10^–5 M indol-3yl acetic acid (IAA)5 x 10^–6 M 6-benzylaminopurine (BA) and 5 x 10^–4Minositol enhanced root thickening, increasing stele and xylemdiameters in roots cultured both with and without attached shoottissues. The effects of shoot tissues and enrichment of themedium were additive. The effects of auxin, cytokinin and gibberellin (gibberellicacid, GA²) were also studied on daxpitated seedlings. BA wasmuch more effective in inducing cell divisions in the hypocotylthan either IAA or GA supplied separately but a mixture of IAA+GAalso produced clearly defined arcs of cambial tissue. Littlesecondary tissue had been produced after seven days' treatment,and stelar enlargement was due to the development of a cambialzone and cell expansion in the primary tissues. Only minor differencesin response were observed between the two varieties. No stimulation of storage organ development occurred when auxin,cytokinin or inositol was inwrporated into the inorganic culturesolution in which plants of Cherry Belle were grown. Rnphanus sarivus, radish, storage organ, cambial activity, growth promoters, indol-3-ylacetic acid, 6-benzylaminopurine, gibberellic acid     

Frond and flower production in Lemna gibba G 3 in presence of respiratory inhibitors

Effects of respiratory inhibitors on frond and flower productionin light culture of a long-day duckweed, Lemna gibba G 3, wereinvestigated. The inhibitors examined could be divided into3 groups based on their specific actions: (A) 2,4-Dinitrophenol(10^–6M), arsenate (10^–4M), malonate (10^–2M),o-phenanthroline (10^–6M), ,'-dipyridyl (10^–5M) andazide (10^–6M) inhibited flower production by suppressingthe rate of flower production without affecting the inductionperiod. Frond production, however, was promoted by these reagents.Effective time of application came one day after the end ofthe induction period. (B) Iodoacetate (10^–6M) and fluoride(10^–4M) inhibited both flower production and, less significantly,frond production. Reduced rate of flower production was responsiblefor the inhibition of flowering. Effective time of applicationpreceded by one day that of A group inhibitors. (C) Salicylaldoxime(10^–6M), diethyldithiocarbamate (10^–6M) and 8-hydroxyquinoline(10^–7M) enhanced flower production by reducing the lengthof the induction period, and simultaneously slightly inhibitedfrond production. Effective time of application was the latterhalf of the induction period. The implications of these findingsare discussed with special reference to the component processesinvolved in photoperiodic induction of flowering in duckweed. (Received March 27, 1969; )     

Some effects of salicylic acid on growth and flowering in Spirodela polyrrhiza SP20

More than 70% flowering was observed in Spirodela polyrrhizaSP₂₀ under long and short days, when fronds were grown in amedium supplemented with 5x10^–5 M salicylate (SA). Toour knowledge this is the first report of such profuse floweringof this duckweed in vitro. Besides flowering, SA also affectedthe multiplication rate, anthocyanin and chlorophyll contents,and gibbosity. Aspirin had an effect similar to that of SA. (Received April 1, 1980; )