Characterization of Melittin Effects in Synaptosomes

The effects of melittin at increasing concentrations on: [³H]GABA release from mouse brain synaptosomes; on the radioactivity released from [³H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 M progressively increases [³H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [³H]GABA induced by melittin below 3 M is Ca²⁺ dependent, but not that induced by the higher concentrations. The Ca²⁺ dependent fraction of the [³H]GABA released by 0.3 M melittin is selectively inhibited by 10 M quinacrine and 1 M nordihydroguaiaretic acid (NDGA) and facilitated by 3 M indomethacin, whereas the Ca²⁺ independent fraction of the [³H]GABA released by melittin is not. In the presence of Ca²⁺, melittin 0.3, 1 and 10 M progressively increases [³H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 M treated synaptosomes, but a noticeable disorganization is produced in 10 M melittin-treated synaptosomes, independently on the presence of external Ca²⁺. LDH activity only increases over control activity in the supernatant solutions of 10 M melittin treated synaptosomes, also in a Ca²⁺ independent manner. Our interpretation of these results is that the Ca²⁺-dependent, pharmacologic sensitive component of melittin-induced release of [³H]GABA, unmasked when 0.3 M melittin was used, involves the activation of a Ca²⁺-dependent type of membrane PLA₂. The Ca²⁺-independent release of [³H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Reciprocal Innervation between Serotonergic and GABAergic Neurons in Raphe Nuclei of the Rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [³H] serotonin or [³H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA_A receptor agonist muscimol (3 to 30 M) and the GABA_B receptor agonist baclofen (30 and 100 M) inhibited [³H]serotonin and [³H]GABA release. These effects of muscimol were reversed by the GABA_A antagonists bicuculline (100 M). The GABA_B antagonist phaclofen (100 M) also antagonized the baclofen-induced inhibition of [³H]serotonin and [³H]GABA release. Phaclofen by itself increased [³H]serotonin release but it did not alter [³H]GABA overflow. Muscimol (10 M) and baclofen (100 M) also inhibited [³H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA_A and GABA_B receptors located on serotonergic neurons. The 5-HT_1A receptor agonist 8-OH-DPAT (0.01 to 1 M) and the 5-HT_1B receptor agonist CGS-12066A (0.01 to 1 M) inhibited the electrically stimulated [³H]serotonin and [³H]GABA release. The 5-HT_1A antagonist WAY-100135 (1 M) was without effect on [³H]serotonin and [³H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 M) did not alter the inhibitory effect of CGS-12066A (1 M) on [³H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 M) to reduce [³H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 M) still inhibited [³H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 M) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT_1A/B and GABA_A/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.     

Three dimensional reconstruction of the X-Y pair during pachytene in the rat (Rattus norvegicus)

The ultrastructure of the X-Y pair from rat spermatocytes has been reconstructed sterically by the study of serial sections. The X-Y pair of the rat at early pachytene contains two dense cores, a long and a short one, which form a synaptonemal complex 1.7 long at their common end. The long core (10.6 ) and the short core (4.5 ) correspond to X and Y, respectively. There is no RNA histochemically detectable in the X-Y pair. Nucleoli are independent of the X-Y pair. A low number of cells show nucleoli very near the X-Y pair but no continuity exists between these structures.     

Aflatoxin influences on seed germination and root elongation by two cultivars of Glycine max and uptake of 65 Zn-ZnCl2 by the cultivars

Maximum inhibition of Glycine max, cv. Essex seed germination occurred at 10 g/ml following 72 hr imbibition in constant light. Seeds imbided 108 hr in constant darkness at this concentration showed a 20% rise in germination over that of the control. Imbibition of G. max, cv. Williams seeds in either light or dark for 96 hr did not suppress germination. Imbibition of Essex seeds in either light or dark at 2.5 through 10 g/ml stimulated root elongation except for 10 g/ml at 96 hr (light). Maximum inhibition of Williams root elongation under constant light was at 48 and 72 hr with 10 g/ml. Statistically significant differences in cotyledon, leaf and stem lengths between non-treated (NT) and treated (T) seedlings were not found except for Williams stem length at 2.5 / ml. Root elongation was stimulated 1.2- and 1.1-folds, respectively, at 5.0 (Essex) and 2.5 (Williams) g/ml. Toxin at 2.5 through 10.0 g/ml did not markedly alter either cotyledon or leaf widths with the exception of Williams leaf width at 2.5 g/ml. Medium supplementation with 2.5 through 10.0 g/ml resulted in cotyledon, leaf and root weight enhancements for Essex seedlings. Stem weight was not markedly affected. An 18% rise in Williams cotyledon weight above that of the control was seen at 2.5 g/ml. Williams leaf weights were increased 1.75- and 1.25-folds, respectively, at 2.5 and 10.0 g/ml. Aflatoxin B₁, at 2.5 g/ml promoted Williams stem and root elongation 1.20- and 1.09-folds, respectively. Most of the radioactivity from ⁶⁵Zn-ZnCl₂ recovered within organs was found within Essex roots for both T and NT seedlings. A higher amount of radioactivity was recovered within roots at each toxin concentration than was without toxin. However, this was not statistically significant. Significant differences in the distribution of radioactivity within roots between NT and T Williams seedlings were not observed. Generally, AFB₁ failed to affect significantly these two varieties of soybeans based on the tests relating to germination, growth and radiolabel uptake.     

Clearance rates of bacteria-sized particles by freshwater ciliates,measured with monodisperse fluorescent latex beads

Summary Monodisperse fluorescent latex particles with diameters of 0.57 and 1.04 m have been used to measure the clearance rates of bacteria-sized particles by two ciliates from a Norwegian lake. The clearance rates by Epistylis rotans on these particles were in the range 0.23 to 1.26 l ind^-1h^-1, and by Strombidium sp. 0.26 to 0.90 l ind^-1 h^-1. The clearance rates varied with food level and temperature. Possibilities and limitations of the suggested method are discussed.     

6-Methylpurine-induced inhibition of sclerotia morphogenesis in Sclerotium rolfsii and its reversal by adenosine

In liquid synthetic medium inoculated with Sclerotium rolfsii (SR), addition of 6-methylpurine (MP, 50g/ml) immediately after inoculation led to approximately 100% reduction in sclerotia production. Adenosine, and to a lesser extent guanosine, each at final concentration of 100g/ml significantly reduced inhibition of sclerotia formation by SR in presence of 50g/ml MP. Uridine and cytidine each at 100g/ml had no such effect. The inhibition of sclerotia morphogenesis could be prevented by addition of 800g/ml of adenosine together with 50g/ml MP. Reversal by adenosine of MP-induced inhibition of sclerotia development was concentration dependent.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

Growth inhibition of biomass adapted to the degradation of toluene and xylenes in mixture in a batch reactor with substrates supplied by pulses

A biomass adapted to degrade toluene and xylenes in mixture was grown in a batch reactor with substrates supplied by pulses. The inhibition of biomass growth in the course of substrate degradation was investigated. The maximal biomass concentration of 7 g l^–1 was obtained using 150 l of toluene and 15 l of a mixture of xylenes in one litre of liquid medium, and the maximal biomass productivity and yield were 53 mg l^–1 h^–1 and 0.32 g_DW g _s ^–1 , respectively. Higher quantities of substrate added by pulses, that is 200 l of toluene with 20 l of xylenes and 300 l of toluene with 30 l of xylenes, caused an accumulation of metabolites. These higher quantities of substrates caused inhibition of microbial growth. Among the metabolites produced, 4-methyl catechol was found in large quantities in the culture medium and in the cells.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Influence of sarmesin on some dopamine-related types of behaviour

Summary The present study was undertaken to investigate the effects of sarmesin, an analogue of [Sar¹] angiotensin II (ANG II) where the tyrosine hydroxyl group in position 4 is methylated, on dopamine (DA)-related paradigms: locomotor and exploratory behaviour as well as apomorphine (3 mg/kg, ip)-induced stereotypy in rats. Sarmesin (0.5 and 1 g, icv) significantly decreased ambulation and rearing movements, and blocked the inhibitory effect of ANG II (0.1 g) on both types of activity. Sarmesin induced biphasic effects on apomorphine-induced stereotypy depending on the dose increase (0.5 and 5 g, icv) and decrease (10 g). Moreover, sarmesin (5 g) blocked the inhibitory effect of ANG II (2 g, icv) on apomorphine stereotypy. Taken together, these results suggest that sarmesin might interact with AT₁ and AT₂ receptor subtypes. The results further confirm the statement for ANG II-DA interaction in brain structures involved in these types of behaviour.     

Epizooic ciliates (Vorticella sp.) compete for food with their host Daphnia longispina in a small polyhumic lake

Summary Clearance rates of epizooic ciliates (Vorticella sp.) were measured together with their host, a planktonic cladoceran Daphnia longispina by using fluorescent latex beads as tracers of food. Vorticellans and their host graze on food of same size range (nanoplanktonic algae and bacteria). Individual clearance rates of Vorticella averaged 6.9 and 7.0 l ind^-1 h^-1 and those of Daphnia 463 and 708 l ind^-1 h^-1 for beads with diameter 2.00 and 3.92 m. On the average, epizooic vorticellans together on the carapace of Daphnia cleared particles with rates representing 25–33% of that the host cleared, the maximum rates being 50–80%. In a steeply stratified polyhumic lake vorticellans take advantage of following Daphnia to food patches and they can severely compete for food with their host.     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity