Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Different effects of two brassinosteroids on growth,auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2,3, 17-trihydroxy-5-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the yes or no type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N⁶-(²-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.Abbreviations BR(s) brassinosteroid(s) - 24-epiBR 24-epibrassinolide - THA-BR 2,33, 17-trihydroxy-5-androstan-6-one - CK(s) cytokinin(s) - iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - Z trans-zeatin - [9R]Z ribosyl-trans-zeatin - ABA abscisic acid - IAA indole-3-acetic acid - NAA naphtalene-1-acetic acid - DEAE cellulose diethylaminoethyl cellulose - HPLC high performance liquid chromatography - ELISA enzyme linked immuno-sorbent assay     

Influence of physical conditions of nutrient medium and sucrose on somatic embryogenesis of date palm

Shoot tips and leafy bud fragments removed from offshoots of adult date palms (Phoenix dactylifera L.) were cultured on a nutrient medium containing the Murashige and Skoog inorganic salts, 453 M 2,4-dichlorophenoxyacetic acid, 14.8 M N⁶-(2-isopentenyl)adenine and 3 g l^-1 activated charcoal to develop nodular callus after 8 months of culture. Callus was cultured in agar-solidified and stationary or shaken liquid media containing half-strength MS inorganic salts, 3 g l^-1 activated charcoal and different sucrose concentrations to study the influence of these factors on somatic embryogenesis. The best conditions for embryo development were culturing in liquid medium shaken at 100 rpm for a period of 2 weeks without sucrose, followed by culture on 3% sucrose.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP N⁶-(2-isopentenyl) adenine - MS Murashige & Skoog (1962) - rpm revolutions per minute     

Cytokinins in cut carnation flowers: I. The complex in ovaries

Tentative identification of the cytokinins present in extracts of Dianthus caryophyllus ovaries using High Performance Liquid Chromatography and Radioimmunoassay techniques, revealed the presence of trans-ribosylzeatin, trans-zeatin, dihydrozeatin and N⁶ (²-isopentenyl)adenine. In addition slow moving compounds (paper chromatography) which could be hydrolysed by -glucosidase were also detected. After hydrolysis the active compounds co-chromatographed with zeatin and ribosylzeatin.     

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A     

Partial auxin deprivation affects endogenous cytokinins in an auxin-dependent,cytokinin-independent tobacco cell strain

The dynamics of individual endogenous cytokinins within the growth cycle (subculture interval) of an auxin-dependent and cytokinin-independent cell suspension culture ofNicotiana tabacum L. (strain VBI-0) were determined using high performance liquid chromatography and radioimmunoassay. In cells grown at an optimum auxin concentration the transient maxima of N⁶-(²-isopentenyl)adenine and N⁶-(²-isopentenyl)-adenosine correlated with the onset of cell division. Cultivation of the cells in a partially auxin-deprived medium resulted in ca. tenfold increase of all endogenous cytokinins. A very distinct maximum of N⁶-(²-sopentenyl) adenine appeared at the beginning of subculture. This indicates that a lack of auxin induced an accumulation of cytokinins predominantly in the form of the free bases, which are physiologically more active than the corresponding ribosides.Abbreviations iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - t-Z trans-zeatin - t-[9R]Z trans-zeatin riboside - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - f.w. fresh weight - SBI subculture interval - C complete medium - PAD partially auxin-deprived medium - RP-HPLC reverse phase high performance liquid chromatography - RIA radioimmunoassay - PAL L-phenylalanine ammonia lyase     

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and -glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.Abbreviations GUS -glucuronidase - HPT hygromycin phosphotransferase - bar phosphinothricin acetyl transferase gene - PPT phosphophinothricin - NAA naphthalene acetic acid - 2iP 2-isopentenyl adenine     

Plant regeneration capacity of callus derived from leaf,stem, and root segments of Populus alba L. x P. grandidentata Michx.

Summary Expiants for establishing callus cultures originated from in vitro cultured hybrid poplar (Populus alba L. X P. grandidentata Michx.). Plant regeneration was achieved from established callus cultures derived from stem internodes (SI), leaf discs (LD), and root segments (RS). Shoot regeneration from callus occurred within 4 weeks of culture on most of the media tested. Frequency of shoot formation was greatly increased by subculturing the selected organogenic calli on regeneration media. The highest rate of multiple shoot formation (an average number of 7/SI, 11/LD, and 8/RS) was obtained by using 0.05 M IBA in combination with 22.5 M 2iP, 22.5 M zeatin, and 12.5 M 2iP, respectively. Regenerated shoots were easily rooted in polyterra peat plugs in transparent plastic boxes. The rooted plantlets were subsequently transferred to pots containing an artificial potting mix.Abbreviations BA 6-benzylaminopurine - 2iP N⁶-isopentenyladenine - Z zeatin - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - WPM Woody Plant Medium - PGR Plant Growth Regulator Journal Paper No. J-14119 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2210     

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Summary Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.Abbreviations BM basal medium - BA 6-benzyladenine - CIM callus induction medium - CPM callus proliferation medium - (diH)Z dihydrozeatin - DW dry weight - ELISA enzyme linked immunoassay - FW fresh weight - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - NAA 1-naphthaleneacetic acid - PBS phosphate buffer - PGR plant growth regulator - (diH)[9R]Z 9--D-ribofuranosyl-(diH) - [9R]iP 9--Dribofuranosyl-iP - [9R]Z 9--D-ribofuranosylzeatin - TBS trishydroxymethyl-aminomethane buffer - Z zeatin     

In vitro establishment and multiplication of Nymphaea hybrid ‘James Brydon’

Rhizome tips were the most suitable explants for in vitro plant regeneration and multiplication of Nymphaea hybrid James Brydon on Murashige and Skoog medium containing different concentrations and combinations of indole-3-acetic acid, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), kinetin, 2-isopentenyladenine (2iP) and gibberellic acid (GA₃). A combination of 2iP, BA, and NAA strongly favored induction of shoot buds and shoot proliferation. Pretreatment of shoot cultures at 8°C for 30 days or with 14.4 or 28.9 M GA₃ for 15 days did not improve shoot multiplication. A 16-h photoperiod with photosynthetic photon flux of 30 mol m^-2 s^-1 was found to be the optimum light condition for shoot growth and multiplication. Multiple shoots produced well developed root systems within 4 weeks after transfer to a plant growth regulator-free medium containing activated charcoal.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid     

Analyse spectrophotométrique du phytochrome dans l'embryon de Courge (Cucurbita pepo) et de Potiron (Cucurbita maxima)

Summary Phytochrome detection and (O.D.) measurements were made for the first time in a single embryo of pumpkin or gourd thanks to an apparatus adjusted to the differential spectrophotometer constructed by Dr. Spruit (Wageningen). Observed (O.D.) values are comparable with usual values obtained by analysis of several seeds in a cuvette. The differential spectrum obtained has the same characteristics as the spectra of seedlings and seeds which can be found in the present literature. The time course of phytochrome appearance in a single pumpkin embryo showed the different phases previously described in lettuce seeds. Use of masks which delimit different areas on the embryo made it possible to locate changes in phytochrome concentration. Analysis of pieces cut off from embryos and packed into a cuvette showed the existence of a decreasing concentration gradient from the cotyledons to the vegetative axis.

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Ce travail a été réalisé au laboratoire de Physiologie des Plantes de Wageningen (Pays-Bas) où nous avons reçu l'hospitalité et les conseils du Dr Spruit. Nous avons bénéficié de la collaboration technique de Mlle D. Eude.     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

Endogenous cytokinins in vegetative shoots of peas

In G2 peas senescence only takes place in long days. In order to determine the role of cytokinins in this process the endogenous cytokinins from vegetative shoots of G2 peas were characterized using gas chromatography-mass spectroscopy following purification by HPLC. Cytokinins were extracted and purified with and without the addition of ¹⁵N labelled internal standards of several cytokinins to estimate cytokin content by isotope dilution in the mass spectra. Samples without internal standards were bioassayed after HPLC. Bioassays showed the presence of zeatin, zeatin riboside and zeatin-0-glucoside. The presence of zeatin was confirmed by its mass spectrum of its permethylated derivative. Tentative identification of zeatin riboside, zeatin-0-glucoside, dihydrozeatin, and dihydrozeatin-0-glucoside was obtained by the coincidence of the major ion for the permethylated natural and ¹⁵N labelled internal standards on GC-MS, and the similar coincidence of ions for permethylated zeatin riboside-0-glucoside by direct probe MS. There was no indication of the presence of significant quantities of zeatin-7-glucoside or zeatin-9-glucoside. The amounts in the tissue ranged from 200–1000 ng/kg fresh weight for each cytokinin and about 2–4 g/kg fresh weight for total cytokinins. There was no apparent difference in the levels in mature but pre-senescent shoots grown in long days and short days indicating that apical senesecence in G2 peas does not appear to be induced by a decline in cytokinin level in the shoots.Cytokinin abbreviations CK Cytokinin - Z trans zeatin - [9R]Z t-zeatin riboside - [9R-5P] Z t-zeatin riboside-5-monophosphate - (OG)Z t-zeatin-0-glucoside - (OG)[9R]Z t-zeatin riboside-0-glucoside - [7Z]G t-zeatin-7-glucoside - [9G]Z t-zeatin-9-glucoside - (diH)Z dihydrozeatin - (diH)[9R]Z dihydrozeatin riboside - iP N6(²-isopentenyl) adenine - [9R]iP N6(²-isopentenyl) adenosine Work performed while PJD was on leave at the University College of Wales at Aberystwyth.     

Preliminary investigations on somatic embryogenesis from leaf discs of red oak (Quercus rubra L.)

Somatic embryos were obtained from leaf discs of juvenile red oak plants. Basal inductive nutrient medium was a modified Murashige and Skoog solution enriched with 500 mg L^–1 casein hydrolysate, 100 mg L^–1 polyvinylpyrrolidone, 5.4 M naphthaleneacetic acid and 0.09 M benzyladenine. Embryogenesis was obtained only from leaf discs in the presence of light and increased when the adaxial surface of the explants (with midrib or main veins present) was in contact with the medium. Large variation was observed in all experiments. Recurrent embryogenesis was observed at the base of embryo clusters with callus present; conversely, embryogenic potential was rapidly lost by subculturing full calli. Maturation, germination and development of isolated somatic embryos were obtained. However, the vast majority of embryos did not have viable apical bud meristems and on only a few occasions were shoots produced.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2iP isopentenyladenine - NAA naphthaleneacetic acid - 2.4-D 2.4-dichlorophenoxyacetic acid - GA gibberellic acid - PVP polyvinylpyrrolidone     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

The differential effect of N6-benzyl-adenine and N6-(Δ2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

Summary N⁶-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N⁶-(²isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.Abbreviations ABA abscisic acid - BA N⁶-benzyl-adenine - BHT butyl-hydroxy-toluene - ELISA enzyme linked immunosorbent assay - FM fresh mass - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - MI mitotic index - 9RBA 9-ß-D-ribofuranosyl-BA - 9RiP 9-ß-Dribofuranosyl-iP - 9RZ 9-ß-D-ribofuranosyl-zeatin - Z zeatin     

Endogenous hormone concentrations and embryogenic callus development in wheat

Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins ₁, ₃ and ₂₀ [GAs], zeatin/zeatin riboside [Z/ZR] and N ⁶[²-isopentenyl] adenine/ N ⁶[²-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)