Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Three cultivars of M. sativa and one cultivar of O. viciifoliawere evaluated for their response to inoculation with A. rhizogenesstrain A4T (containing pRiA4b). A cultivar-dependent responsewas observed in M. sativa with 94%, 25%, and 4% of infectedstem explants producing transformed roots in the cultivars Vertus,Regen-S, and Rangelander, respectively. In O. viciifolia cv.Hampshire Giant, an explant-dependent response was observedwith 78% and 50% of seedling cotyledon and hypocotyl explantsresponding, respectively. Leaf explants failed to produce transformedroots. Transformed roots showed plagiotropic and negativelygeotropic growth on hormone-free agar MS medium. Productionof transgenic shoots from O. viciifolia root cultures occurredspontaneously. Recovery of transgenic plants from M. salivacv. Rangelander was achieved by transfer of callus (inducedon UM medium containing 2·0mg dm^–3 2,4-D and 0·25mg dm^–3 kinetin) to MS medium containing 0·5 ingdm^–3 BAP and 0·05 mg dm^–3 NAA. Cultured rootsof both species synthesized opines (agropine and mannopine).Extensive morphological variation was observed in plants ofM. sativa (clone Al) and O. viciifolia (clone A4Tl) establishedin the glasshouse. DNA sequences homologous to TL-DNA and TR-DNAwere present in root clones and regenerated plants. Key words: Agrobacterium rhizogenes, Medicago sativa, Onobrychis viciifolia, transformed roots, transgenic plants     

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     

Scanning Electron Microscope Studies of Multiple Shoot Production by Meristem Tip Cultures of Solarium x curtilobum

A meristem tip culture isolated from Solatium x curtilobum cv.Mallku produced a limited amount of callus at the cut surfaceof the explant when cultured on Murashige and Skoog medium containing1 mg dm³ 6--{dimethylallylamino)purine and O.01 mg dm³ naphthaleneacetic acid. A single sheet of cuticle-like material develops on localizedregions of the callus surface and the cells beneath it decreasein mean cell size. These meristematic centres develop into fullyorganized shoot meristems, and each will grow out into a leafyshoot when the culture is transferred to growth medium containingo.1 mg dm³ gibberellic acid (GA₃) as the only growth hormone.This system has considerable value in the rapid clonal propogationof Solanum species, since as many as fifty shoots can be producedfrom a single meristem tip culture.     

Phenotypic Effects of Isolated pRiA4 TL-DNA rol Genes in the Presence of Intact TR-DNA in Transgenic Plants of Solanum dulcamara L.

The effect of the rol genes, together with the TR-DNA of pRiA4on the phenotype of Solanum dulcamara plants, was analysed.Plants transformed by Agrobacterium strain BN1010: :rolA (rolA^7plus;TR⁺)exhibited severe leaf wrinkling, whereas plants transformedby strain BN1010: :rolC (ro/C⁺TR⁺) had a typical ‘hairyroot’ phenotype. Leaf discs excised from these latterplants produced roots on hormone-free medium. BN1010: :rolABC(rolABC⁺TR⁺) transformed plants had an exaggerated transformedphenotype. Some of the BN1010: :rolABC transformants had positivelygeotropic root growth which correlated with the presence ofmultiple copies of the TR-DNA. S. dulcamara plants, transformedby the TR-DNA region only, exhibited epinasty. Scanning electronmicroscopy of plants containing various regions of agropineRi T-DNA revealed that transformation causes changes in basicplant siructure.     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

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     

Influence of nutrients on the development of gibbosity in fronds of the duckweed Lemna gibba L.

The duckweeds Lemna gibba L. and Lemna minor L. only grew wellin undisturbed culture under axenic conditions in low lightintensity when provided with a suitable energy source such asglucose. In media containing N0₃-N gibbosity (a convex ventralsurface) was induced in the presence of the chelating agentethylene-diamine-di-o-hydroxyphenylacetic acid (EDDHA). In nutrientsolutions containing NO₃-N as the only N source, but withoutEDDHA, L. gibba occasionally exhibited gibbosity in culturesolutions of 40 cm³ volumes. More fronds were induced to exhibitgibbosity when the volume of the culture medium was increasedfrom 40 cm³ to 200 cm³. Gibbosity was never induced in L. minor,neither was it induced in L. gibba in media containing NH₄-N,even in the presence of NO₃-N. There was no direct correlationbetween the occurrence of gibbosity and frond growth rate, butgibbosity occurred only when there was good frond growth. In the absence of a sugar, frond growth was enhanced by bubblingair through the culture solution in the light. Increasing theCO₂ concentration in the air up to 1% enhanced growth and inducedgibbosity. Carbon dioxide did not induce gibbosity in mediacontaining NH₄-N. Key words: Ammonium-N, carbon dioxide, gibbosity, Lemna, nitrate-N     

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     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

LA ENPRENO DE STRONTIO EN KOKOLITOFOROJ

Pure cultures of the coccolithophorid Syracosphaera carleraewere grown in a synthetic saline medium containing 3.4mM Ca++and 2.0, 1.0, 0.5 or 0.0 mM Sr⁺⁺. The coccoliths were separatedby differential centrifugation, washed, dried, and examinedby flame photometry and by X-ray diffraction. In the absenceof Sr, they consisted of pure calcite. In media containing Sr,the concentration factor or incorporation factor (Sr/Ca in coccolithsSr/Ca in medium) was approx. 0.02 in each case, indicating ahigh degree of discrimination against Sr. ¹ Nuna adreso: Scripps Instituto por Oceanografio, La Jolla,California, U. S. A. (Received March 4, 1961; )     

Effect of Light and NaCI Salinity on the Growth of Callus Cultures of Ipomoea pes-caprae (L.) R. Brown and Ipomoea batatas (L.) Lam

Callus cultures of Ipomoea pes-caprae and I. batatas were establishedon MS medium containing 10^–5 M 2,4-D and 10^–8 Mbenzyladenine. Ipomoea pes-caprae calli exhibited green pigmentationand grew better in the light than in darkness. Callus tissuesof I. batatas showed a pale-yellow colouration and they grewat the same rate in light as in dark conditions. I. pes-capraeand I. batatas callus cultures were sensitive to the presenceof 60 mM NaCl in the culture medium, the growth of the formerbeing more sensitive in light than in darkness. The significanceof the responses of I. pes-caprae callus cultures in relationto the mechanism of salt tolerance is discussed. Ipomoea batatas, Ipomoea pes-caprae, sweet potato, railroad vine, callus cultures, salinity, light     

Reversible Structural Changes Associated With Callus Formation and Plantlet Development from Aseptically Cultured Shoots of Taro (Colocasia esculenta var antiquorum)

Taro callus maintained on Knop's medium with 2, 0·2 or0·02 mg l^–1 2,4,5-trichiorophenoxyacetic acid (2,4,5-T)or Linsmaier-Skoog (LS) containing 1 mgl^–1 of the cytokininadenine-N-benzyl-9-tetrahydro-2H- pyran-2-yl (SD8339) or 6 dimethylaininopurineand 0·1 mgl^–1 -naphthaleneacetic acid underwenta transition to a stable organized growth form which is referredto as a calloid. On transfer to LS medium th 0·2 mgl^–12,4,5-T in the absence of cytokinin the calloid reverts backto callus. Colocasia esculenta(L.)Schott, taro, callus, calloid, in vitro selection, histology, micropropagation, tissue culture, cytokinin     

Effect of NaCl Salinity on Somatic Embryo Development in Sapindus trifoliatus L.

The effect of NaCl salinity on growth and development of somaticembryos of Sapindus trifoliatus L. was examined. Incorporationof 25 and 50 mol m^–3 NaCl into the medium greatly increasedthe growth and development of somatic embryos and both theseconcentrations favoured the production of secondary embryoids.However, supplementation of 100 mol m^–3 NaCl to the mediumdid not have any significant effect on the growth and developmentof somatic embryos. On the other hand, the culturing of proembryostructures in medium containing 200 mol m^–3 NaCl resultedin complete death within 7 d of salt exposure. Analysis of somatic embryos revealed that, upon salinization,they accumulated Na⁺ and Cl^– in significant amounts butthe content of Na⁺ was much less compared to that of Cl^–.Addition of NaCl (up to 50 mol m^–3) in the medium resultedin a considerable increase in the K⁺ content of somatic embryos.The content of proline in somatic embryos, however, increasedsubstantially in response to salinization. The amount of freesterols, steryl glycosides, steryl esters, and phospholipidsalso rose to higher values in salt-affected somatic embryos.The results suggest that somatic embryos of S. trifoliatus cantolerate concentrations of NaCl up to 100 mol m^–3 withoutaffecting growth and that they have sufficient cellular mechanismsto tolerate salinity at relatively high levels. Key words: Salinity, somatic embryo, sterols, phospholipids     

Production of Plantlets of Shorea roxburghii G. Don. from Embryonic Axes Cultured In Vitro

Development of axillary shoots was induced in embryonic axesof the dipterocarp Shorea roxburghii G. Don. cultured on a modifiedMS medium containing 6-benzyl-aminopurine (BAP) at an optimumconcentration of 5 mg I^–1. Excised axillary shoots wereused in multiplication and rooting experiments. Vigorous rootdevelopment occurred in shoots supported on filter paper bridgesin liquid medium containing naphthaleneacetic acid (NAA) andindolebutyric acid (IBA) (0.1 mg I^–1 each). Shorea roxburghii, Dipterocarpaceae, tissue culture, plantlet formation     

CHANGES IN HYDROGENASE ACTIVITY DURING THE SYNCHRONOUS GROWTH OF SCENEDESMUS OBLIQUUS D3

1. A fairly good synchronization of Scenedesmus cells was obtainedby transferring the cells grown in a medium containing a lowconcentration of iron into a medium containing relatively highconcentration of iron.
2. During the synchronous culture in themineral medium, a goodparallelism between the average cellvolume and hydrogenaseactivity was observed.
3. Effect of glucoseon the development of the hydrogenase activitywas variabledepending on the stage of algal growth.
4. Iron is essentialfor the development of the hydrogenase activityand glucosesupplementary.

¹On leave from Laboratory of Applied Botany, Faculty of Agriculture,Kyoto University, Kyoto.     

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     

Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet Cultured In Vitro

Detrez, C., Tetu, T., Sangwan, R. S. and Sangwan-Norreel, B.S., 1988. Direct organogenesis from petiole and thin cell layerexplants in sugar beet cultured in vitro.—J. exp. Bot.39: 917–926. Plant regeneration was obtained by direct bud formation frompetiole as well as from thin cell layer explants taken fromsugar beet (Beta vulgaris L.) plants grown in vitro. The budswere mainly induced in the blade-petiole transition zone ofthe explants. High frequency bud regeneration was observed inpetiole and thin layer explants of 10 different breeding linesof sugar beet tested. Organogenesis resulted when petiole explantsexcised from 8-d-old seedlings grown on half-strength Murashigeand Skoog medium (MS) containing 3.0 mg dm^–3 naphthaleneacetic acid (NAA), 3.0 mg dm^–3 6-benzylaminopurine (BAP)and 1.0 mg dm^–3 2, 3, 5, triiodobenzoic acid (TIBA) werecultured on MS with 3.0 mg dm^–3 NAA and 3.0 mg dm^–3BAP. Thin cell layer strips isolated from shoot apices culturedon MS medium supplemented with 0–9 mg dm^–3 BAP or1.0 mg dm^–3 indolebutyric acid (IBA) formed adventitiousbuds on MS medium containing 0–5 mg dm^–3 NAA + 5.0mg dm^–3 BAP. Histological studies confirmed the sub-epidermalorigin of shoots. Key words: Beta vulgaris, direct organogenesis, in vitro culture, petiole, regeneration, thin cell layer     

Seed Water Relations and the Regulation of the Duration of Seed Growth in Soybean

Soybean [Glycine max (L.) Merrill] seeds and cotyledons weregrown in an in vitro culture system to investigate the relationshipsbetween cell expansion (net water uptake by the seed) and drymatter accumulation. Seeds or cotyledons grown in a completenutrient medium containing 200 mol m^–3 sucrose continueddry matter accumulation for up to 16 d after in planta seedsreached physiological maturity (maximum seed dry weight). Seedor cotyledon water content increased throughout the cultureperiod and the water concentration remained above 600 g kg^–1fresh weight. These data indicate that the cessation of seeddry matter accumulation is controlled by the physiological environmentof the seed and is not a pre-determined seed characteristic.Adding 600 mol m^–3 mannitol to the medium caused a decreasein seed water content and concentration. Seeds in this mediumstopped accumulating dry matter at a water concentration ofapproximately 550 g kg^–1. The data suggest that dry matteraccumulation by soybean seeds can continue only as long as thereis a net uptake of water to drive cell expansion. In the absenceof a net water uptake, continued dry matter accumulation causesdesiccation which triggers maturation. Key words: Glycine max (L.) Merrill, solution culture, duration of seed growth, water content, dry matter accumulation     

Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

Dependency of H+ efflux on ATP in cells of Chara australis

The internodal cell of Chara australis was made tonoplast-freeby internal perfusion with a medium containing a Ca²⁺-chelatorEGTA and the net H⁺ efflux across the plasma membrane was estimatedeither by titration of the external medium or by measuring thechange in pH in the external medium. The amount of H⁺ effluxwas high in the presence of internal ATP and low in its absence.The ATP-dependent net H⁺ efflux was about 40 nmoles/m²/sec.This amount is smaller than that estimated for the pump currenton the basis of electrical data obtained earlier (3). This discrepancyis discussed. (Received June 18, 1980; )