Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Mechanism of interferon action: inhibition of pppA(2'p5'A)n-dependent ribonucleases activity in micrococcal nuclease-treated mouse L cell-free extracts

The ability of nonpreincubated as compared to micrococcal nuclease-treated mouse L cell-free extracts supplemented with 2′-deoxythymidine-3′, 5′-diphosphate (pTp) and ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) to catalyze the 2′,5′-oligoadenylate-dependent degradation of reovirus [³H]mRNA was investigated. 2′,5′-Oligo A tetramer enhanced degradation in nonpreincubated but not micrococcal nuclease-treated extracts. Neither pTp nor EGTA significantly affected the 2′,5′-oligo A-dependent degradation in nonpreincubated extracts. The presence of both micrococcal nuclease and calcium was required to establish the subsequent reduction in both 2′,5′-oligo A-dependent and -independent degradation observed in micrococcal nuclease-treated extracts containing pTp and EGTA.     

INFLUENCE OF CERTAIN PLANT GROWTH REGULATORS AND CROWN-GALL RELATED SUBSTANCES ON BUD FORMATION AND GAMETOPHORE DEVELOPMENT OF THE MOSS PYLAISIELLA SELWYNII

Bud formation and gametophore development were studied in the moss Pylaisiella selwynii (Kindb.) Steere and Anderson grown from spores in a liquid medium consisting of inorganic salts. Indoleacetic acid and ethrel increased bud formation within a narrow concentration range. Copious bud formation was obtained with the five cytokinins tested at concentrations varying from 5 X 10⁻⁶ to 5 X 10⁻¹⁴ M. Except for about 10 % of the buds obtained with 6-γ, γ-dimethylallylaminopurine at 5 times 10⁻¹⁴ m, the cytokinin-induced buds failed to develop into normal gametophores. Octopine, lysopine, and octopinic acid, substances obtained from crown-gall tumors, increased bud formation at 10⁻³ m. On lysopine-treated plants these buds developed into typical gametophores. Gemma-like structures were obtained with octopine but no gametophores. l -arginine and l -lysine, the amino acids which respectively occur in octopine and lysopine, failed to induce gametophore formation although buds were obtained with 10⁻³ m lysine. γ-Guanidinobutyric acid induced bud formation at 10⁻³ m, but these buds developed into highly abnormal gametophores. The failure of buds obtained with many of these treatments to develop into gametophores appeared to result from the formation of new cell walls in other than the normal geometrical relationship during initial divisions of the pro-bud. The relevance of the findings to the crown-gall problem is discussed.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening

The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanol-boiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-section up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems.Abbreviations EGTA ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid - PEx Instron plastic extensibility     

Localization of calcium on the stigma surface of Ruscus aculeatus L.

By use of chlorotetracycline and X-ray microanalysis it is demonstrated that the receptive surface of the stigma of Ruscus aculeatus is rich in calcium. The high level of calcium is found in the epidermal cells and in the exudate covering the stigma. These results indicate that in vivo, as in vitro, calcium takes part in the regulation of pollen grain germination.Abbreviations CTC chlorotetracycline - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Effects of prostaglandin D2 on membrane potential in neuroblastoma X glioma hybrid cells as determined with a cyanine dye

A cyanine dye, diS-C₃-(5) was used to determine the effects of prostaglandins on the membrane potential in neuroblastoma X glioma cells (NG 108-15). The largest depolarization was seen with prostaglandin D₂ (ED₅₀ = 1.5 μM), and relative potencies of various prostaglandins (3 μM) were: D₂, 100; I₂, 41; E₁, 17; E₂, 7; and F_2α, 7. 5-Hydroxytryptamine in a dose over 100 μM also depolarized the membrane. The effect of prostaglandin D₂ was observed in a Na⁺-free medium or when Ca²⁺ was replaced by Sr²⁺. The addition of 3 mM ethylene-glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or 5 mM Co²⁺ partially inhibited the effects. These observations suggest that the depolarization of membrane by prostaglandin D₂ may primarily be related to alteration of Ca²⁺ permeability in the cell membrane.     

Toxoplasma gondii: Calcium lonophore A23187-mediated exit of trophozoites from infected murine macrophages

The Ca²⁺ ionophore A23187 consistently induced the exit of Toxoplasma gondii trophozoites from cultured macrophages which they had recently infected. Following exit of toxoplasmas, the host macrophages underwent degeneration. A23187 was active at concentrations higher than 0.25 μM and the activity reached a plateau at the concentration of 1.0 μM. Noninfected macrophages or those engulfing heat-killed toxoplasmas, or some other particles, were not affected by treatment with A23187. The toxoplasmas exiting host cells were capable of infecting and proliferating in normal macrophages. The A23187-mediated exit of toxoplasmas proceeded despite external Ca²⁺ and was enhanced by the addition of ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) in the reaction mixture. On the other hand, the A23187-mediated exit of toxoplasmas was inhibited significantly by exogenous Mg²⁺.     

Activation of Cl− channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca²⁺ concentration ([Ca²⁺]₀) were examined in freshly isolated rabbit osteoclasts. K⁺ current was suppressed by intracellular and extracellular Cs⁺ ions. In this condition, high [Ca²⁺]₀ evoked an outwardly rectifying current with a reversal potential of about −25 mV. When the concentration of extracellular Cl⁻ ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4′,4-diisothiocyanostilbene-2′,2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl⁻ channels. Cd²⁺ or Ni²⁺ caused a transient activation of the Cl⁻ current in contrast to the sustained activation elicited by Ca²⁺. Intracellular 20 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl⁻ current. Either when the osmolarity of extracellular medium was increased, or when 100 μM cAMP was dissolved in the patch pipette solution, high [Ca²⁺]₀ still elicited the Cl⁻ current, indicating that the divalent cation-induced Cl⁻ current was carried through Ca²⁺-activated Cl⁻ channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl⁻ current, indicating that the activation of Cl⁻ current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl⁻ channels under whole cell clamp. Intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) activated the Cl⁻ current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl⁻ current was mediated through a G protein, and that an increase in [Ca²⁺]_i was critical for the activation of Cl⁻ channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl⁻ current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca²⁺-activated Cl⁻ channels. © 1996 Wiley-Liss, Inc.     

What's new: To boldly glow…. Applications of laser scanning confocal microscopy in developmental biology

The laser scanning confocal microscope (LSCM)

1 LSCM: laser scanning confocal microscope; FISH: fluorescence in situ hybridisation; DiO₆: 3,3′-dihexyloxacarbocyanine iodide; NBD-ceramide: 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-caproyl)sphingosine; DiO: 3,3′-dioctadecyloxacarbocyanine perchlorate; DiI: 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate; CCD: charge-coupled device; DIC: differential interference contrast; FURA2: (-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy)-2-)2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid, sodium salt);BCECF: 2′,7′-bis-(carboxyethyl)-5-(and-6-)-carboxyfluorescein;fluo-3: 1-(2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-(2′amino-5′-methylphenoxy)-ethane-N,N,N′,N′,-tetraacetic acid, ammonium salt; DAPI: 4′,6-diamidino-2-phenylindole, dihydrochloride; PET: positron emission tomogrophy; CT: computer-assisted tomogrophy; CiD: cubitus interruptus dominus; MRC: Medical Research Council; TOTO-1: benzothiazolium-4-quinolinium dimer; YOYO-1: benzoxazolium-4-quinolinium dimer; ex.: excitation wavelength; em.: emission wavelength.

is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.     

The role of hydroxyl radicals and calcium ions in the priming of a respiratory burst in neutrophils and the increase in luminol-dependent blood chemiluminescence on exposure to combined magnetic fields with a very weak low-frequency alternating component

The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA AM) used at low concentrations (1.0 and 2.5 μM) was shown to block the priming effect of weak combined static (42 μT) and low-frequency collinear alternating (1.0, 4.4, and 16.5 Hz; 0.86 μT) magnetic fields. This blockage was inferred from a greater increase in chemiluminescence observed for a mouse neutrophil suspension exposed to combined magnetic fields in response to the bacterial peptide N-formyl–Met–Leu–Phe added in the presence of luminol. Similar results were obtained for the effect of BAPTA AM on luminol-dependent chemiluminescence of whole blood. The priming effect of weak combined magnetic fields on the respiratory burst in neutrophils did not depend on the presence of extracellular Ca²⁺ and was not affected by the hydroxyl radical scavenger dimethyl sulfoxide used at 0.025–1.0 mM.     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

Intracellular calcium and skin tumor promotion: Calcium regulation of the induction of epidermal ornithine decarboxylase activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin explants maintained in a serum-free Eagle's HeLa cell medium. Chelation of extracellular calcium by ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Extracellular magnesium could not replace calcium for ODC induction by TPA. Concurrent incubation of skin pieces with a calmodulin inhibitor trifluoperzine (TFP) inhibited ODC induction. Furthermore, inclusion in the medium of lanthanum, which has a higher affinity for calcium-binding sites than calcium and displaces surface-bound calcium, inhibited ODC induction by TPA.     

Promotion of Pylaisiella selwynii growth and gametophore formation by octopine and cytokinin

Growth, branching and gametophore formation by protonema developing from spores of the moss Pylaisiella selwynii were promoted by octopine, an unusual amino acid found in crown gall tumors. Cytokinin (0.01 μ M ) in combination with octopine (0.1 μ M to 1 m M ) increased the number of gametophores formed and decreased the time required for their development. The combined effect of these two compounds was similar to that obtained with virulent agrobacteria. At higher cytokinin concentrations, protonemal buds formed abnormal masses of cells rather than normal gametophores. Common amino acids and auxins, alone or in combination with cytokinin, had relatively little effect on the development of the moss.     

Cytokinins in Populus×robusta: Changes during Chilling and Bud Burst

Cytokinin levels in sap and vegetative buds of Populus×robusta Schneid have been determined during chilling and bud burst. From non-detectable levels in December and January, parallel increases in cytokinin levels occur in sap and buds during February and March, both in material from the field and that held at 2°C in the dark. The maximum in the sap occurs two weeks prior to natural bud burst, and 3 weeks, prior to the maximum attained in the buds. Excised twigs, forced to bud burst, show a similar pattern. The role of roots as a possible source of cytokinins is discussed. Partition chromatography on Sephadex LH-20 indicates that at least 5 cytokinins are present in buds, two of which have similar elution volumes to zeatin and zeatin riboside-The main activity in the sap is confined to a zeatin riboside-like component.     

Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (～50%) respond to 100 μM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 μM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.     

The role of wall calcium in the extension of cell walls of soybean hypocotyls

Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozenthawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxy-methyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues. This research was supported by Department of Energy grant DE-FG06-88ER13830 and NASA grant NAGW 1394. The authors are grateful to Dr. David Rayle (San Diego State University, Cal.) for stimulating discussions and comments during the course of this work.     

Activation of a pea membrane protein kinase by calcium ions

Membranes from the buds of Pisum sativum L. contain a protein kinase which is activated 5- to 15-fold by micromolar levels of calcium. Best calcium activations were found with light-membrane fractions, and on density gradients these band at a similar position to the plasma membrane. Other heavier membranes, however, also contain a calcium-dependent protein kinase. The activity of the calcium-dependent protein kinase is inhibited by added phospholipids and phospholipase, in contrast to protein-kinase C. Calcium-dependent protein-kinase activity can be inhibited by 40% by low concentrations of the calmodulin inhibitor, trifluoperazine, but inhibitions are detected only after prior incubation of the membranes for some hours in ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid. Substantial calcium-dependent protein-kinase activity remains uninhibited by trifluoperazine indicating that there may be calmodulin-dependent and calmodulin-independent, but calcium-activated, protein kinases in pea membranes. The calcium-activated protein kinase seems to be intrinsically bound to membranes and only slight or partial solubilisation is obtained by the detergents nonidet P-40, (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate or octyl glucose. Better solubilisation is obtained by acetone treatment. There is some retention of calcium activation after partial solubilisation. A calcium-independent protein kinase has also been detected in membrane preparations; it has a substrate specificity different from that the calcium-dependent enzyme. Our results indicate, therefore, that there may be at least three protein kinases attached to pea shoot membranes.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - TFP trifluoperazine     

A method for isolation of cytoplasmic RNA from a slime mold,Physarum polycephalum

A procedure for fast and simple preparation of cytoplasmic ribonucleic acid from Physarum polycephalum microplasmodia is described. Microplasmodia are homogenized in a high-magnesium-high-ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid buffer and nuclei are pelleted. The supernatant is extracted with sodium dodecyl sulfate-phenol-chloroform and crude RNA is precipitated. This is further purified by selective ethanol precipitation from 6 m guanidinum hydrochloride. This RNA preparation is suitable for direct use in hybridization studies.     

Micropropagation of Guazuma crinita mart. by root and petiole culture

Summary Shoot organogenesis of Guazuma crinita Mart. from root and petiole explants was obtained via adventitious bud formation. Root segments and petiole explants excised from in vitro generated plantlets were cultured on woody plant medium (WPM) supplemented with [trans-6-(4-hydroxy-3-methylbut-2- enyl)aminopurine] (zeatin) or with [6-benzyladenine] (BA). After 45 d of culturing, clumps of green bulbous structures containing small adventitious buds (clusters) were generated in all explants cultured with 10 μM zeatin under a photon flux density of 65 μmol m⁻² s⁻¹. For subsequent shoot differentiation, clusters were transferred onto medium containing 1 μM zeatin. After 60 d of culturing, 30% of clusters generated from petiole explants developed into plants. The regenerated plantlets were successfully acclimatized and all survived and grew well. No morphological abnormalities were observed.