Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Ethylene and Ethane Production in 2,4-D Treated and Salt Treated Tobacco Tissue Cultures

Gas chromatography was used to measure ethylene (ethene) andethane production by tobacco (Nicotiana tabacum cv. Wisconsinno. 38) callus tissues grown on media containing inorganic saltsaccording to Murashige and Skoog (1962), sucrose, myo-inositol,thiamine-HCl kinetic according to Linsmaier and Skoog (1965),and either 2,4-dichiorophenoxyacetic acid (2,4-D) in the range0–100 mgl^–1 or 2 mgl^–1 indoi-3-ylacetic acidplus NaCl in the range 0–200 Meq l^–1. Ethylene productionrates were high (> 500 nl h^–1 g^1– fresh weight)initially in all treatments. Subsequently, ethylene productiondeclined in rapidly growing cultures but remained high in moderatelyand severely 2,4-D (> 0·5 mgl^–1) stressed andin severely NaCl (150 Meql^–1) stressed cultures. Highinitial rates of ethane production (> 200 nl h^–1 g^–1fresh weight) were obtained under conditions of severe stresscaused by 2,4-D or NaCl but not in control or moderately inhibitedcultures. With further incubation ethane production declinedin the severely stressed cultures. It is concluded that ethyleneproduction can be used as an index of moderate 2,4-D stressand severe NaCl stress by virtue of the high persisting ratesof ethylene production in stressed cultures. Ethane productioncan be used as an early index of severe stress caused by either2,4-D or NaCl in vitro. Nicotiana tabacum L., tobacco, ethylene, ethenen, ethane, 2,4-dichlorophenoxyacetic acid, auxin, stress, callus tissue     

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associatedwith a large accumulation of glucose and fructose in the vacuolesof the fruit cells. These hexoses are derived from sucrose,which is released from the phloem and may be taken up by parenchymacells prior to hydrolysis. We have expressed two putative ripening-relatedsucrose transporters from grape berries, VvSUC11 (synonymouswith VvSUT1) and VvSUC12, in an invertase deficient yeast strainto characterize their transport activities. Sucrose was takenup by yeast transformed with either transporter at an optimumpH of <4.5 and with a Michaelis constant (K_m) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibitedby protonophores and by vanadate. This is consistent with anactive uptake mechanism involving proton cotransport, typicalof sucrose/H⁺symporters. The transporters from grape berrieswere functionally similar to Scr1, a sucrose transporter fromRicinus cotyledons. It is likely that in grape berries VvSUC11and VvSUC12 facilitate the loading of sucrose from the apoplastinto the parenchyma cells. Copyright 2001 Annals of Botany Company Fruit, grape berries, plasma membrane, sugars, sucrose transporters, Vitis vinifera     

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 and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

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     

Intracellular Localization and Properties of NADH-Glutamate Dehydrogenase from Vitis vinifera L.: Purification and Characterization of the Major Leaf Isoenzyme

Glutamate dehydrogenase was partially purified from grapevine(Vitis vinifera L. cv. Soultanina) tissues and its activityand isoenzymic pattern were studied. Seven anodal migratingisoenzymes were revealed after PAGE. Leaf protoplasts were isolatedfrom in vitro-grown axenic shoot cultures and used to studythe intracellular localization of GDH. Results revealed thatthe enzyme was associated with the mitochondrial fraction. Theisoenzyme with the lowest electrophoretic mobility, which accountedfor 35 to 40% of total activity, was purified 2050-fold to homogeneityfrom leaves. The purification method included ammonium sulphatefractionation, DEAE-cellulose chromatography, Sephadex G-200gel filtration and NAD-sepharose affinity chromatography. Themolecular weight of the native enzyme was estimated to be 252kDa and it consisted of identical 42.5 kDa subunits. pH optimumfor the aminating reaction was 8.0 and for the deaminating reaction9.3. At optimum pH conditions the apparent K_m values for ammonium,as ammonium chloride and ammonium sulphate, -ketoglutarate,NADH, glutamate, and NAD⁺ were 45.0, 13.0, 2.1, 0.069, 18.0,and 0.195 mM, respectively. The amination reaction of GDH wasfully activated with about 100 µM Ca²⁺ while the deaminationreaction was not affected by the addition of Ca²⁺. The isoenzymesof GDH showed different magnitude of their activating responseto calcium ions. Key words: Vitis vinifera L., glutamate dehydrogenase     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

Modification of Leaf Formation by Cytokinin and Chlormequat (CCC) in Vitis

Application of the cytokinin, 6-(benzylamino)-9-(2-tetrahydropyrany1)-9H-purine(PBA, 1mM) and 2-chloroethyl trimethyl ammonium chloride (chlormequat;3 mM), a growth retardant, to grapevines induced the formationof fused leaves (two laminae and two petioles) and double leaves(two separate petioles each with a single lamina). Double leaveswere found in Vitis vinifera L. seedlings, in Vitis ripariaand in Muscadinia (Vitis) rotundifolia. In some of the treatedvines, leaves arose with opposite phyllotaxy. Other anomaliesincluded production of two opposite axillary buds in axils ofopposite leaves and production of two tendrils per node. Sometendrils grew into shoots. In Muscat of Alexandria, applicationof GA₃ (3 µM, 15 µM), followed by an applicationof chlormequat (0.4 mM, 1.2 mM), led to a marked reduction inthe extension growth of axillary shoots. Vitis vinifera L., Vitis riparia, Muscadinia (Vitis) rotundifolia, grapevines, leaf formation, cytokinin, chloromequat     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

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     

Afternoon Depression In Photosynthesis in Grapevine Leaves--Evidence for a High Light Stress Effect

A midday depression in net photosynthesis and in stomatal conductancewas observed when leaves of well-watered Vitis vinifera plantswere subjected to a diurnal pattern of variation in leaf temperatureand leaf-to-air water vapour pressure difference similar toa summer day, while photon flux density was kept constant at1450 µmolm^–2 s^–1,. When leaves were kept atconstant leaf temperature (22.5°C) and leaf-to-air watervapour presure difference (8.5 Pa kPa^–1) at the same lightintensity, stomata opened with the onset of illumination andmaximal conductance and photosynthesis values were observedabout 1 h later. Subsequently, conductance and photosynthesisdecreased gradually. Leaf water potential never dropped below{macron}0.3 MPa. Leaves kept under constant environmental conditionsshowed an afternoon decline in photosynthesis at high internalCO₂, in carboxylation efficiency and in maximum conductanceas well as an increase in stomatal sensitivity to CO₂. Whenthe photon flux density during the day was reduced to 750 µmolm^–2 s^–1, the afternoon depression in gas exchangerates was attenuated. To evaluate the possible effects of highlight stress on changes in chloroplastic behaviour we comparedlight response curves of photosynthesis determined with an oxygenelectrode, in the morning and in the afternoon after the plantswere exposed to either high or moderate photon flux densities.A significant depression in photosynthetic capacity was foundby this method in high light treated leaves, but not in leavespreviously exposed to moderate photon flux density. Apparentquantum yield decreased in the afternoon, particularly afterexposure to high light. Maximum chlorophyll a fluorescence at22°C was reduced and the quenching of fluorescence afterreaching the peak was slower in the afternoon than in the morning,especially in high light-treated leaves. Changes in the patternsof chlorophyll fluorescence kinetics were observed after lighttreatment, i.e. in the afternoon, with oscillations either absent(after high light) or significantly reduced (after moderatelight) in comparison to the morning. The significance of theseresults is discussed and it is suggested that a direct inhibitoryeffect of high light at the chloroplast level provides the bestinterpretation for the observed afternoon decline in photosyntheticrate. Key words: Carboxylation efficiency, chlorophyll fluorescence, photosynthetic capacity, quantum yield, stomatal conductance, Vitis vinifera L.     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Embryonic Organogenesis

Somatic embryos ofVitis vinifera L. ‘Grenache noir’develop abnormally and form viable plantlets at very low frequencies.They grow continuously and, after the torpedo stage, they formgiant structures which do not undergo further organogenesis.Morphological, histological and cytochemical data were usedto study development from the globular to the giant-embryo stage.Histological organization of somatic embryos until the torpedostage was similar to that of zygotic embryos. Somatic embryosformed bipolar axes, which differentiated precociously and simultaneouslya root and a shoot meristem. However, they differed from theirzygotic homologues by forming a cotyledonary crown or multiplecotyledons and by their rapid cellular differentiation. At theend of the torpedo stage and up to the giant-embryo stage, somaticembryos underwent some characteristic events of germination:the radical grew, tannins accumulated, and protodermal cellssuberized. However the shoot apex was rapidly disorganized anddisappeared. This peculiar behaviour is discussed in comparisonwith the phenomenon of precocious germination often observedfor immature zygotic embryos inin vitro culture. Vitis vinifera ; grapevine; somatic embryo development; precocious germination; histology     

Growth and Development of Roots of Grapevine (Vitis vinifera L.) in Relation to Water Uptake from Soil

Developmental patterns of lateral roots and their vascular differentiationwere investigated for Vitis vinifera L. cv. Shiraz to assessthe likely contribution of lateral roots to total water uptakeof plants subjected to different irrigation regimes. Correlationanalyses showed a significant positive correlation between mainroot diameter and the diameter of first order lateral rootsof well-watered plants, but in water-stressed plants the twowere not significantly correlated. The correlations betweendiameters of first order lateral roots and the diameters ofmain roots were greater than correlations between the lengthsof first order laterals and the diameters of main roots. Thesuberised surface area of well-watered main roots increasedfrom 4% of total surface area at 0·25 cm to 100% at 10cm from the tip, whereas that of stressed plants increased from15% at 0·25 cm to 100% at 5 cm from the tip. In all treatmentsthe highest linear density of first order laterals was about7 laterals cm^-1 of main root. More than 50% of first order lateralshad diameters less than 0·05 cm, and more than 90% ofthem had lengths less than 5 cm. Calculations of axial resistancesbased on xylem diameter measurements suggest that the axialresistances of root segments may not be uniform along rootsas is often assumed in models of water uptake. Water flow intothe main roots via the lateral root pathway is likely to bemuch smaller than that via the direct radial flow pathway asonly about 1% of surface area of main roots is directly occupiedby lateral roots, leaving the other 99% of main root surfacearea available for the direct radial flow pathway.Copyright1994, 1999 Academic Press Axial resistance, grapevine (Vitis vinifera L. cv. Shiraz) roots, root diameter, root length, xylem vessels     

Somatic Embryos and Plantlets from an Ancient Clone of the Grapevine (cv. Cabernet-Sauvignon) by Apomixis in vitro

Plantlets were produced by induction of nucellar embryony (apomixis)in isolated unfertilized ovules of a non-apomictic plant—thegrapevine (Vitis vinifera L. cv. Cabernet-Sauvignon). Ovulesgrown in liquid culture with benzyladenine (5–10 µM)and ß-naphthoxyacetic acid (5–25 µM) formeda nucellar callus which gave rise to somatic embryos. For growthof plantlets the embryos were transferred to an agar mediumcontaining gibberellic acid (1 µM) and 2-isopentenyladenine(5 µM). This is the first report of somatic embryo formationin a cultivar of a temperate fruit.     

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     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid