Promotion of Plant Cell Division by an Epidermal Growth Factor

In some specified treatments, an epidermal growth factor (EGF)promoted adventitious root formation in epicotyl cuttings ofVigna angularis. The number of the roots induced in cuttingstreated with 0.1 mg liter^-1 EGF during the first 24 h and with210^-4 M IAA during the second 24 h was 15% greater than thatof the roots in cuttings treated without EGF and with IAA. Analysisof the optimum timing of EGF application was performed by dividingthe first 24 h period into three sequential 8 h periods (0–8h, 8–16 h and 16–24 h). The most effective timeperiods in terms of the root formation were 8–16 h and16–24 h. The 0–8 h period was ineffective with respectto the formation. When carrot suspension cells were culturedfor 15 days at a very low cell density (1,000 cells/3 ml Murashigeand Skoog's medium) with more than 0.1 mg liter^-1 EGF, cellnumbers were 72% higher than those cultured without EGF. Theseresults suggest that EGF promotes cell division of plants. (Received October 5, 1992; Accepted May 24, 1993)     

Effects of Boron Deficiency on the Chemical Composition of a Marine Diatom

Cells of Cylindrotheca fusiformis, a marine pennate diatom,multiplied in complete nutrient medium (containing 0?5 mg l^–1B) with a generation time of 11 h; in a boron-deficient culturemedium (0?02 mg l^–1 B), the generation time was 90 h.The chemical composition of logarithmically growing cells harvestedfrom each of the two culture conditions was compared in orderto assess the effects of boron deficiency on the various chemicalfractions. Under conditions of boron deficiency, the concentrationof protein, carbohydrate, and RNA was depressed below that ofcontrol cells; DNA showed no change, while lipids, phenoliccompounds, and unaccounted-for organic fractions were all increasedin concentration. The greatest effect was on the phenolic fraction.These results are discussed and related to the published literatureon effects of boron deficiency on higher plants.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Mineral nutrition and auxin-induced growth of Triticum coleoptile sections

A revised method has been described for assaying auxin by thegrowth of Triticum coleoptile sections. With additions of Ca(NO₃)₂10^–4 and MgSO₄ 10^–5 mol liter^–1 the sensitivityand accuracy have been increased. This is mainly due to Ca.The coleoptiles obviously suffer from Ca-deficiency. The importanceof a strict time schedule for manipulations is emphasiced. Theduration of the tests is limited to 6 hr.Indole-3-acetic acidcan be determined in concentrations down to 10^–9mol liter^–1;from 10^–10 to 10^–9mol liter^–1 the log-logrelation between concentration and growth is linear. Above 10^–7mol liter^–1 elongation takes place under an abnormal increasein elastic extension, indicating that growth is limited by someunknown wallstabilizing factor. The interrelation between auxin,an anti-auxin and Ca are discussed. (Received May 8, 1973; )     

COMPARATIVE LIFE-CYCLE, BIOMASS AND SECONDARY PRODUCTION OF THREE SYMPATRIC FRESHWATER GASTROPOD SPECIES

The life cycles, biomass and secondary production of three sympatricfreshwater basommatophoran snails, Lymnaea palustris (MÜller),Physa fontinalis Linnaeus and Anisus rotundatus (Poiret) werestudied during two years in a freshwater ditch. L. palustrisexhibited an iteroparous life-cycle whereas the two other speciespresented a semelparous life-cycle, adults died just after oviposition.L. palustris secondary production (dry weight) value was higher(P = 11 298.4 mg 0.1 m^–1 yr^–1) than those of P.fontinalis (P = 846.3 mg 0.1 m^–2 yr^–1) and A. rotundatus(P = 1192 mg 0.1 m^–2 yr^–1). (Received 16 March 1992; accepted 30 June 1992)     

Effects of an Epidermal Growth Factor on Growth of Zea Primary Roots and Mesocotyls

Sections of the elongation zones of primary roots and mesocotylsof Zea mays L. were incubated with various concentrations ofhuman epidermal growth factor (EGF). The growth rates of rootsections incubated with 1,000, 100 and 10 µg liter^–1EGF were higher than that of control by 15%, 26% and 14%, respectively.The rates of mesocotyl sections incubated with 100, 10, 1.0and 0.1µg liter^–1 EGF were 23%, 31%, 24% and 22%higher than that of control. (Received August 8, 1994; Accepted November 1, 1994)     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H₃BO₃), Na⁺, and Cl^– fluxes. Neither Na⁺ nor Cl^– appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent K_m 1–2 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H⁺ gradient. Addition of 30 mM HCO₃^– does not affect boron extrusion by Bor1p, indicating that HCO₃^– does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H₃BO₃ causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w     

Effect of External Supply of Growth Substances on Axillary Proliferation and Development in Dioscorea bulbifera

Bulbil development in cultured nodes of D. bulbifera proceededin the absence of growth substances from the medium. When IAAwas incorporated into the medium at the concentrations of 5mg l^–1 and 10 mg l^–1 the cultured nodes producedlarger bulbils than in its absences. When the concentrationof IAA was increased to 15 mg l^–1, however, the culturednodes produced a callus instead of a properly organized bulbil.The dry weight of bulbils increased when kinetin was added tothe medium at the concentrations of 0.05, 0.5, and 2.5 mg l^–1.The greatest increase was with 0.5 mg l^–1 kinetin. Onincreasing the concentration of kinetin in the medium to 5.0mg l^–1 the tissue produced had smaller dry weight thanthose produced in the absence of growth substances. Additionof different combinations of IAA and kinetin to the basal mediumresulted in the production of normal bulbils, roots, and shootsin some instances (suitable combinations) and in the productionof callus and abnormal shoots in others (non suitable combinations).     

Effects of Glycollate on the Growth of a Planktonic Chlorella

The duration of the lag phase in cultures of a planktonic strainof Chlorella pyrenoidosa growing from small inocula under conditionsof light limitation was shortened by the addition of 1 mg 1^–1of glycollate to the medium. Of eight related compounds tested,none, with the possible exception of lactate, had a similareffect. Addition of 1 mg 1^–1 of glycollate also considerablyincreased the relative growth-rate of the alga at low (500 lux)but not at higher light intensities. This effect was not, however,specific to glycollate. Concentrations of glycollate (20 mg1^–1) higher than those normally produced in cultures byexcretion had an inhibitory effect on growth.     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

Somatic Embryogenesis and Its Hormonal Regulation in Tissue Cultures of Freesia refracta

Somatic embryogenesis can be induced in tissue cultures of Freesiarefracta either directly from the epidermal cells of explants,or indirectly via intervening callus. These two pathways ofsomatic embryogenesis can be controlled and regulated by varyingthe combinations and levels of exogenous hormones. When younginflorescence segments were cultured in vitro on modified N₄(MN₄) medium supplemented with 2 mg l^–1 indoleacetic acid(IAA) and 3 mg l^–1 6-benzylaminopurine (BAP), some ofthe epidermal cells began to exhibit the features of embryogeniccells. These cells produced embryoids and developed into newplants through direct somatic embryogenesis. If the same explantswere placed on Murashige and Skoog's (MS) medium containing2 mg l^–1 IAA, 05 mg l^–1 BAP and 05 mg l^–1naphthaleneacetic acid (NAA), pale-yellow translucent nodularcalluses appeared on the surface of the explants. When thiskind of callus was transferred to MN6 medium with 2 mg l^–1IAA and 3 mg l^–1 BAP, embryoids formed which further developedinto plantlets. The regenerated plants were morphologicallynormal and possessed the normal diploid chromosome number of2n = 22. A similar result has also been obtained with youngleaf explants of this plant. The early segmentations of embryogeniccells and the development of embryoids were studied using histologicaland scanning electron microscopic techniques, and the resultshave been discussed in association with the ontogeny and originof the embryoids. Freesia refracta Klatt, somatic embryogenesis, plant regeneration, exogenous hormones     

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     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture     

Micropropagation of Pissardi Plum

An efficient medium for multiplication of Prunus cerasifera,J. F. Ehrh. cv. ‘Atropurpurea’, Pissardi plum, consistedof Linsmaier-Skoog basal medium (LS) supplemented with 3% sucrose,10mg 1^–1 N⁴-(²-isopentenyl) adenine (2iP), and 162 mg1^–1 phloroglucinol (Phl). Phl in the medium significantlyenhanced growth (P < 0.1 %) over cultures maintained on LSmedium with 10 mg 1^–1 2iP and no Phl. Plantlets were rootedon a half-strength Murashige-Skoog (MS) medium supplementedwith 0.2 mg 1^–1 indole-3-butyric acid (IBA). Prunus cerasifera, Pissardi plum, micropropagation, phloroglucinol, N⁶-(²-isopentenyl) adenine     

Callus Culture of Sainfoin (Onobrychis viciifolia) and Plant Regeneration through Somatic Embryogenesis

Callus of sainfoin (Onobrychis viciifolia Scop.) was initiatedfrom stem and root explants which were obtained from seedlingsgrowing in vitro, on Linsmaier Skoog (LS) medium supplementedwith 1 mg l^–1 2, 4-D and 1 mg l^–1 BA or only 1 mgl^–1 BA, and the Vacin and Went medium without hormones.Somatic embryos were formed on LS medium containing 1 m l^–1BA. Embryos developed into complete plants on filter paper saturatedwith hormone-free LS medium. Onobrychis viciifolia, somatic embryogenesis, callus culture, plant regeneration     

Somatic Embryogenesis in Cassava: The Anatomy and Morphology of the Regeneration Process

Anatomical and morphological studies demonstrated that somaticembryos developed similarly on mature seed and clonal leaf explantsof cassava (Manihot esculenta Crantz) cultured for 20–24d on Murashige and Skoog (MS2) basal medium supplemented with4.0 mg l^–1 2,4-D (Stage 1) before transfer to MS2 basalmedium supplemented with 0–01 mg l^–1 2,4-D and 0–1mg l^–1 6-benzylaminopurine (Stage II medium). Within 7d of inoculation onto Stage I medium, cell divisions occurredin the adaxial tissues of cotyledon-piece and leaf-lobe explants,and associated with this was the development of embryogeneticprotusions and ridges on the adaxial surface. Foliose structuresand somatic embryo initials developed from these tissues oncotyledon, embryonic axis and leaf-lobe explants and, when cultureswere transferred to Stage II medium, further somatic embryodevelopment occurred. Somatic embryos apparently originatedfrom groups of cells and were identified by the presence ofa closed root axis, a shoot axis and cotyledons of similar shapeand venation to those of zygotic embryos. Somatic embryos hadno vascular connection with parental cultures. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, anatomy, morphology, morphogenesis     

Photoautotrophy of Spinach Cells in Continuous Culture: Photosynthetic Development and Sustained hotoautotrophic Growth

Photosynthetic development of Spinacia oleracea L. (spinach)cells was ‘triggered’ by halving the supply of sugarto a continuous culture. This was demonstrated between two steadystates at constant specific growth rate (4.2 ± 0.17 x10^–3 h^–1). As a direct consequence of this ‘trigger’the cells were in conditions of sugar famine, demonstrated byorganic carbon balance. Comparison of the biomass was made betweenthe two steady states. It was found that in sugar famine thebiomass contained 8 times as much chlorophyll and the ratioof photosynthesis to respiration in saturating conditions hadincreased 22 times, when compared to the biomass in conditionof sugar excess. All substrate carbon and organic factors were then removed fromthe fresh medium, carbon dioxide and photosynthetically activeradiation (PAR) were increased. In these photoautotrophic conditionsthree further steady states were established. The effects ofincreasing the dilution rate and then increasing PAR were examined.Growth was found to be limited by PAR. The photosynthetic yield Y of photoheterotrophic spinach cellsin fructose famine was estimated as 0.004 g kJ^–1 PAR.In photoautotrophic conditions the maximum Y was 0.0018 g kJ^–1.It was suggested that this lower yield was due to chloroplastdevelopment being arrested by an unknown factor.     

In Vitro Morphogenesis in Nardostachys jatamansi DC.: Shoot Regeneration from Callus Derived Roots

Callus cultures of Nardostachys jatamansi DC. maintained onMurashige and Skoog's medium containing 3.0 mg 1^–1 of-naphthaleneacetic acid and 0.25 mg 1^–1 of kinetin whenshifted to medium containing 0.25–1.0 mg 1^–1 ofindole-3-acetic acid or indole-3-butync acid showed profuserhizogenesis. The callus-regenerated roots when transferredto medium containing 2.0–6.0 mg 1^–1 of kinetin producedshoot buds. The de novo shoot bud regeneration took place eitherdirectly from cortical cells or from the inner stelar region.In addition, direct, concomitant root-shoot development wasalso observed. Nardostachys jatamansi, organogenesis, root-buds     

Isolation, Culture, and Plant Regeneration of Protoplasts of Two Shrubby Oxalis Species from South America

Protoplasts were successfully isolated from internodal callustissues of both Oxalis glaucifolia and O. rhombeo-ovata whenthey were digested in a solution containing 0.1% (w/v) MacerozymeR-10, 0.5% (w/v) cellulase Onozuka R-10 and 0.3 mmol m^–3sucrose. Protoplasts proliferated to give cell colonies on Gamborget al.'s B5 medium supplemented with 0.3 mmol m^–3 mannitol,0.5 mg dm^–32, 4-D, and 2.0 mg dm^–3 kinetin. Calluswas produced upon transfer of cell colonies to Murashige andSkoog medium containing 2.0 mg dm^–3 l-naphthaleneaceticacid (NAA) and 0.1 mg dm^–3 kinetin for O. glaucifolia,or with 5.0 mg dm^–3 NAA and 0.5 mg dm^–3 6-benzylaminopurine,for O. rhombeo-ovata. Plants were regenerated from O. glaucifoliaprotoplasts on a medium containing 0.1 mg dm–3 NAA, 1.0mg dm^–3 kinetin and 1.0 mg dm^–3 gibberellic acid,but only vascular nodules were differentiated by O. rhombeo-ovataprotoplast-derived calli. Key words: Tissue culture, protoplasts, plant regeneration, Oxalis spp