Mechanism of a Synergistic Effect of Kinetin on Auxin-induced Ethylene Production: Suppression of Auxin Conjugation

In hypocotyl segments of mung bean (Phaseolus mungo L.) seedlings, exogenously supplied indoleacetic acid was rapidly conjugated mainly into indoleacetylaspartic acid, which was inactive in inducing ethylene production. Kinetin is known to stimulate indoleacetic acid-induced ethylene production. The mechanism of kinetin action on indoleacetic acid-induced ethylene production by hypocotyl segments of mung bean seedlings was studied in relation to indoleacetic acid uptake and indoleacetic acid metabolism. Kinetin enhanced indoleacetic acid uptake during the initial 2-hour incubation and markedly suppressed the conversion of indoleacetic acid to indoleacetic acid conjugates throughout the whole 7-hour incubation. As a result, there was more free indoleacetic acid and less conjugated indoleacetic acid in the segments treated with kinetin than in those receiving no kinetin. A close relationship was demonstrated between the rate of ethylene production and the level of free indoleacetic acid, which was regulated by kinetin.     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

Inhibitory effect of some plant growth regulators,phenolics and fungicides on the production of pectin methylgalaturonase byAlternaria solani andA. tenuis

Secretion of pectin methylgalacturonase (PMG) byAlternaria solani was suppressed in the following sequence of active agents (100 ppm): kinetin > indolebutyric acid > vanillin > gibberellic acid > ferulic acid > 3-hydroxybenzaldehyde > phloroglucinol > indolepropionic acid > indoleacetic acid. InA. tenuis the sequence was kinetin=gibberellic acid=indoleacetic acid=indolebutyric acid (all completely inhibitory) > phloroglucinol > indolepropionic acid > ferulic acid. Of ten fungicides tested, most potent in both species were thiram, brassicol, blimix and sultaf (at 1000 ppm). Mycelial growth was relatively less affected by the active agents and fungicides inA. tenuis (range of % inhibition from 10 to 21) than inA. solani (range from 25 to 67%). There was no clear correlation between the inhibitory effect of the various agents on PMG secretion and on mycelial growth.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Callus induction and shoot regeneration in Sempervivum tectorum

Leaf and shoot explants of Sempervivum tectorum L., taken from 14- and 30-day-old plants germinated in vitro, have been studied by using Murashige-Skoog and White basal media with cytokinins (benzyladenine, kinetin) and auxins (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid) in various concentrations. Explants taken from 14-day-old plants died but 30-day-old leaves and shoots produced yellow and soft, as well as green and hard calluses on Murashige-Skoog medium with 4.4–8.8 M benzyladenine and 0.57 M indoleacetic acid. Shoot organogenesis was induced from green, hard callus in a medium with 2.2 M benzyladenine plus either 1.1 M indoleacetic acid or 2.5 M indolebutyric acid. Whole plants were grown on Murashige-Skoog medium without plant growth regulators. On the other hand, White medium was not suitable for raising Sempervivum tectorum in vitro.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige-Skoog - NAA napthaleneacetic acid - W White     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Effects of genotype,explant source and growth regulators on organogenesis in carnation callus

For callus induction, shoot tips and nodal or internodal stem segments of carnation cultivars (Coral, Jaguar, Salome and Sarinah) were grown on MS basal medium with 2,4-dichlorophenoxyacetic acid and kinetin. To achieve organogenesis, calli were transferred onto MS medium without or with growth regulators (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid, kinetin, benzyladenine) in different combinations. Shoot primordia emerged from the subsurface meristemoids of calli, roots developed from the inner callus cells. The effects of genotype, explant source and growth regulators on callus-mediated organogenesis differently manifested themselves in caulogenesis and rhizogenesis, respectively. The number of root-forming calli most of all depended on genotype and least of all on explant source. Unlike rhizogenesis, caulogenesis essentially depended on explant source: internodal calli of all the tested cultivars practically missed the shoot formation ability. The number of caulogenetic calli from apical-nodal segments significantly depended on genotype, but was also affected by growth regulators. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non-reproductive tobacco plants was tested on a hormone-free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR- and TL-DNA from Agrobacterium rhizogenes 1855 root-inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone-free medium, indoleacetic acid alone (1 μ M ), kinetin alone (1 μ M ), and most abundantly on indoleacetic acid plus kinetin (1 μ M each). No flower formation was observed on indolebutyric acid plus kinetin (10 μ M and 0.1 μ M , respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone-free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.
The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non-reproductive plants.     

THE EFFECTS OF GIBBERELLIC ACID AND A GROWTH RETARDANT ON OVULE FORMATION AND GROWTH OF EXCISED PISTILS OF NIGELLA RANUNCULACEAE

The effects of gibberellic acid (GA₃) and the growth retardant AMO-1618 on ovule formation in excised pistils of Nigella sativa L. were studied by sterile culture techniques. Gibberellic acid promoted pistil growth and inhibited ovule formation. The role of endogenous gibberellins in ovule formation and pistil growth was investigated by adding AMO to the basal medium. Both pistil lengths and ovule formation were reduced significantly with increasing concentrations of AMO. The addition of low concentrations of GA₃ to the medium restored pistil growth but did not reverse the inhibitory effect of AMO on ovule formation. The addition of kinetin or indoleacetic acid (IAA) to the medium containing AMO had no effect on pistil lengths. However, with the addition of 10⁻⁷ m kinetin, the number of ovules in pistils was increased but not to the levels found in pistils grown in the absence of AMO.     

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium Communicated by S. Gleddie     

Ethylene enhances shoot formation in cultures of the peach rootstock GF-677 (Prunus persica x P. amygdalus)

The influence of ethylene on shoot formation from GF-677 (Prunus persica × P. amygdalus) shoot tip explants was studied in vitro. Cultures in test tubes were placed inside 5 1 glass jars and supplemented with various ethylene concentrations (0–10 ppm). Ethylene at 0.1 ppm, applied during the first 2 weeks of culture, increased the number and the length of shoots produced in vitro. Test tubes with cultures sealed with various types of closure accumulated in their atmosphere different levels of ethylene ranging from 0.1 to 1.2 ppm, depending on the type of closure. Test tubes with cotton-wool bungs had the least while those with serum stoppers had the highest amount of ethylene. The maximum number of shoots was recorded in test tubes covered with serum stoppers. The ethylene concentration was related linearly (R=0.974) to the shoot number and exponentially (R=0.975) to the shoot length.Abbreviations BA benzyladenine - IAA indoleacetic acid - HSD honestly significant difference     

In vitro culture and plant regeneration of large flowered purslane

Culture conditions were established for callus induction from a range of Portulaca grandiflora Hook tissues. Rapidly growing calli were obtained on Murashige and Skoog medium with stem-, leaf- and sepal-derived explants. Plant regeneration via organogenesis was explant-origin dependent with hypocotyl tissues giving the highest shooting frequency. Light conditions, pH and carbon source had a pronounced effect on the percentage of explants regenerating buds and the number of buds formed. It was possible to establish stable regenerated plants in the glasshouse.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - MS Murashige and Skoog (1962) medium     

The Effects of Cytokinins on Unfolding of Cotyledons and Opening of Hypocotylar Hooks of Cucumber

Benzyladenine (BA), zeatin and kinetin caused the unfolding of cucumber (Cucumis sativus L. Var. Jing-ye No. 4) cotyledons. But the other plant hormones e.g. gibberellic acid (GA3), indoleacetic acid(IAA), ethylene (ethrel), abscisic acid (ABA) and other related compound e.g. KCl, adenine were not so. This response was caused by even lower concentration (0.01 ppm). Therefore, it could be used as a bioassay for cytokinins. KCl affaeted synergistically on cytokinin-induced unfolding. Basally applied BA (10 ppm) stimulated the opening of the intact hypocotylar hook. The light affacted synergistically on BA-induced opening. However, basally applied BA prevented the opening of decapitated hooks, especially in the light. The cotyledons were found to play a part of effects in maintaining the hook.     

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.     

Clonal propagation of mature elite trees of Commiphora wightii

Elite trees of Commiphora wightii (Arnott) Bhandari were selected from the wild on the basis of their content of guggul, an oleoresin. The selected tree was micropropagated through forced axillary branching on Murashige and Skoog's (MS) medium supplemented with benzyladenine (BA) and kinetin. Highest frequency of shoot formation was achieved on MS medium supplemented with 17.8 M BA, 18.6 M kinetin, 100 mg l^-1 glutamine, 10 mg l^-1 thiamine HCL and 0.3% activated charcoal. Seasonal changes affected the shoot proliferating potential of the initial explants in vitro. Transfer of shoots to a medium containing a lower concentration of BA (1.8 M) and kinetin (1.9 M) before rooting markedly stimulated shoot elongation. Shoots could be rooted by treating them with both indoleacetic acid and indolebutryic acid for 24 h in darkness and transferring them to a low-salt basal medium with activated charcoal. After rooting, transfer to a half-strength White's (modified) medium was necessary for further development of the plantlet. Regenerated plantlets were successfully established in soil.Abbreviations AC activated charcoal - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - 2-iP isopentenyladenine - MS Murashige and Skoog's medium - NAA -naphthaleneacetic acid - PG phloroglucinol     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.