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     

Some Physiological Responses of Chlorella pyrenoidosa to 2,4-Dichlorophenoxyacetic Acid

An 18-h treatment of synchronously-grown Chlorella pyrenoidosawith 2,4-D did not significantly alter the size, dry weight,degree of synchrony, or pigment content of the cells, nor weredetectable quantities of ethylene produced. When Chlorella pyrenoidosawas treated with 5?10^–4 M 2,4-D, there was a statisticallysignificant stimulation of both net oxygen uptake and productionwhile 5?10 M 2,4-D inhibited both processes. When Chlorellapyrenoidosa was treated with 5?10^–4 M and 5?10^–3M 2,4-D, significantly greater amounts of glycollate were presentin the culture medium, even though an assay for glycollate dehydrogenaseshowed that the activity of this enzyme from 2,4-D-treated Chlorellapyrenoidosa was three times greater than in control cells. Looselybound 2,4-D was partitioned from a nonaqueously isolated chloroplastfraction, while other cell fractions failed to show detectablequantities of 2,4-D. It is postulated that in Chlorella pyrenoidosathe chloroplast is a target for 2,4-D action and that interferencein photorespiratory processes may underlie the observed responses.     

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     

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     

Factors Affecting Growth and Aggregate Dissociation in Batch Suspension Cultures of Datura innoxia (Miller)

Growth kinetics of Datura innoxia batch suspension cultureswen monitored by a Klett-turbidimetric technique. While cultured. wt varied linearly with Klett units, f. wt and packed cellvolume did not. Turbidimetrically determined doubling timeswere highly reproducible. The method proved to be useful inthe determination of acutely lethal conantrations of a seriesof anti-metabolites. In certain circumstances, aggregate dissociation in batch suspensioncultures of D. innoxia was found to be coupled to growth rate.Suspensions maintained with 10^–5 M 2,4-D exhibited a relativelyslow growth rate with a high degree of aggregate dissociation:10^–4 M 2,4-D promoted a maximum growth rate, but dramaticallysuppressed aggregate dissociation. At 10^–5 M 2,4-D, themitotic index of smaller-aggregate fractions was greater thanthe mitotic index of the large-aggregate fraction. At 10^–5M 2,4-D the converse was observed. Supraoptimal 2,4-D concentrationsthus enhanced both aggregate dissociation and the growth ofsmaller aggregates. When present in concentrations promoting optimal growth. malicand succinic acids caused a decrease in aggregate dissociation.Casein hydrolysate dramatically enhanced growth, but did notaffect aggregate dissociation to the same degree as 2,4-D orthe Krebs cycle organic acids. Suggestions are made concerningmedium composition to be used in future mutant selection schemesusing D. innoxia. Datura innoxia (Miller), suspension culture, growth kinetics, mitotic index, 2,4-dichorophenoxy acetic acid     

The Effects of 2,4-Dichlorophenoxyacetic Acid on Ion Uptake by Maize Roots

The effects of the synthetic auxin and herbicide 2,4-dichlorophenoxyaceticacid (2,4-D) on K^$ and Cl^– uptake and H^$ release by youngexcised maize roots has been studied. Brief exposure to 2,4-D(0.01 mmol dm^–3) at pH 3.5 causes a large depolarizationof the electrical potential across the root plasma membranesand converts K^$ uptake to K^$ leakage into the bathing solution.These results can be explained by the increased H^$ permeabilityof the membranes induced by the weak acid 2,4-D. The depolarizationresults in a less favourable electrochemical potential gradientfor K^$ uptake across these membranes. These effects are notrelated to the auxin properties of 2,4-D as the nonauxin 3,5-dichlorophenoxyaceticacid (3,5-D) gives rise to similar effects. The relative depolarizationsinduced by a range of weak acids appear to be unrelated to theiroil/water partition coefficients. In contrast, on bathing the roots for longer periods in solutions(pH > 5) containing 2,4-D (0.01 mmol dm^–3) K^$ and Cl^–uptake and H^$ release are inhibited. These effects are not shownwith 3,5-D suggesting an auxin-linked action for 2,4-D. Alsothe electrical potential across the plasma membranes is onlyslightly depolarized so that a change in the electrochemicalpotential gradient cannot be invoked to explain the loweredion fluxes. The evidence is consistent with the removal of anenergy supply to a metabolically linked K^–/H^– exchangemechanism in the plasma membranes. It is likely that both modes of action would operate to lowerion uptake under soil-grown conditions, the former becomingmore manifest in acidic soils.     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Morphogenic Potential of Cultured Explants of Crocus chrysanthus Herbert. cv. E. P. Bowles

Explants of leaves, basal plates, petals, anthers and ovariesof young growing corms of Crocus chrysanthus var. E. P. Bowleswere cultured on MS basal media with 20 different combinationsof either kinetin and NAA or BAP and 2, 4-D in the dark. Nomajor change was observed except on ovary explants. The ovaryexplants produced callus at 5.0 mg 1^–1 and 10 mg^–1BAP and subsequently stigma-like structures formed on the surfaceof the callus. Transfer to light resulted in the stigma-likestructures developing a yellow pigmentation whereupon they cameto resemble the naturally-grown stigmas. Corm formation andshoot regeneration was obtained from the callus when the ovaryexplants were cultured on media containing 5.0 and 10 mg I^–1BAP with 0.5 mg 1^–1 2, 4-D. Increasing the level of 2,4-D markedly reduced the number of shoots produced per explant. Key words: Crocus chrysanthus, callus, ovary explants     

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     

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     

A Preliminary Study on the Effects of Nitrogen Supply on the Growth In Vitro of Nine Potato Genotypes (Solanum spp)

Cultures of nine potato genotypes (seven Solanum tuberosum oneS. sparsipilum and one S. oplocense genotypes) were examinedfor their response to growing on medium containing either 60mol m^–3, 40 mol m^–3 or 20 mol m^–3 nitrogen.Genotypes differed in their response to nitrogen. Reducing thenitrogen regime tended to produce taller plants with longerinternodes, shoots had larger leaves but contained less chlorophyll.No change in fresh weight or number of nodes was observed. Genotypex nitrogen interactions were significant for chlorophyll content,shoot length and internode length. Results suggest that thechanges observed were as a result of changes in the total nitrogenlevel rather than changes in the ammonium : nitrate ratio. Thisstudy suggests that for certain potato genotypes, nitrogen levelsin MS medium are too high for producing desirable microplantsin terms of leaf area and shoot length Key words: Solanum tuberosum, S. sparsipilum, S. oplocense, micropropagation, morphogenesis     

Organogenesis in the Cultured Female Gametophyte of Ephedra foliata

The female gametophyte of Ephedra foliata was used as an explantfor the production of haploids as it is composed of haploidcells, all of the same genotype. The regeneration of roots wasdependent upon the presence of NAA, while BAP had a modifyingeffect. At lower concentrations (0.05 parts 10^–6 and 3.5parts 10^–6) BAP enhanced the root promotion of NAA (0.05–4.0parts 10^–6). At higher concentrations of BAP (1–6parts 10^–6), roots and shoot buds were formed. Kinetinat 4.0 parts 10–6 with 0.5 parts 10–6 2, 4-D wasoptimal for shoot bud production in explants at the archegonialstage and 2, 4-D at 2.0 parts 10–6 with 0.5 parts 10–6kinetin was optimal for root formation. Cells of the callusand root tip had the haploid number of chromosomes, n = 7. Meristemoidswere located on the surface or embedded in the callus tissue.The deep seated meristemoids organized only root primordia,but the peripheral ones gave rise to root as well as shoot budprimordia. Initially, there was no vascular connection betweenthe shoot-bud and the callus. This was established later. Key words: Ephedra, Female gametophyte, Haploid, Tissue culture     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Production of Potato Spindle Tuber Viroid (PSTV) by Large Scale Fermentation of PSTY-Infected Potato Cell Suspension Cultures

Berlin, J., Wray, V., Forche, E., Reng, H.–G , Schler,H, Luckinger, R. and Mhlbach, H.–P. 1985. Production ofpotato spindle tuber viroid (PSTV) by large scale fermentationof PSTV–infected potato cell suspension cultures.—J.exp. Bot 36: 1985–1995. Cell suspension cultures of Solatiumdemissum, infected with the potato spindle tuber viroid (PSTV),were scaled up to volumes of up to 800 dm³ to provide sufficientand uniform plant material for subsequent studies on viroidbiosynthesis. Here we describe the technological aspects ofproducing the required amounts of biomass and viroid. The cells,which had been maintained on a medium containing expensive coconutmilk, were first adapted to rapid growth on the less expensiveB5–medium. The physiological state of the cells was monitoredby in vivo ³¹P–NMR spectroscopy Under the chosen conditionsthe scale–up from 10 dm³ inoculum from shake flasks tothe harvest of the 800 dm³ stirred fermenter lasted 38 d andprovided 112 kg biomass. Growth characteristics and viroid productionin shake flasks and large bioreactors were rather similar. Gelelectrophoretic analysis of isolated nucleic acids using silverstaining and Northern blot hybridization revealed a PSTV–contentof approximately 700 µg PSTV per kg fresh mass of culturedcells. Key words: Solanum demissum, plant cell cultures, potato spindle tuber viroid, biomass production, fermentation, in vivo ³¹P-NMR     

Anthocyanins in Modern Roses: Chemical and Colorimetric Features in Relation to the Colour Range

With respect to intravarietal variability, the petals of 15rose (Rosa x hybrida) varieties, representative of the colourrange expressed by modern roses primarily pigmented with anthocyanins,were investigated from chemical and. colorimetric viewpoints.Depending on the variety, the observed colour variations werebased on a more or less complex mixture of cyanidin 3,5-diglucoside,pelargonidin 3,5-diglucoside, quercetin and kaempferol glycosides.The total anthocyanin content ranged from 4 to 109 mg g^–1petal dry wt., while the total amount of flavonol glycosideswas never less than 8 mg g^–1 and could reach 136 mg g^–1petal dry wt. Between cultivars, the pH of the petal outer epidermisvaried from 3·6 to 5·4 units. Using a spectrocolorimeter,the petal colour of each variety was measured. In order to allowquantitative comparisons of colours, the reflectance curveswere further translated into indices calculated using the CIELabsystem. In the aggregate, there were good correlations betweenchemical parameters and colorimetric indices that are lightness(L^*), chroma (C^*) and hue angle (h). Both of these criteria(chemical and colorimetric) appeared sufficient to explain thevisual sense of the petal colour. Key words: Rose, colour, flavonoid, colorimetry, CIELab system     

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     

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     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Effects of Auxin and Gibberellin on Elongation of Young Hyphae in Neurospora crassa

IAA, 2,4-D and GA₃ promoted the elongation of young hyphae inNeurospora crassa at the optimum concentrations of 10^–6,10^–6 and 10^–4 M, respectively. The effects of IAAand GA₃ were additive. (Received June 17, 1983; Accepted December 22, 1983)     

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