Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Novel prebiotic systems: Nucleotide oligomerization in surfactant entrapped water pools

Summary Oligomerization of 5-TMP in water pools entrapped by dodecyl-ammonium chloride surfactant aggregates in benzene: hexane in the presence of dicyanodiimide at temperatures ranging from 21°–72° resulted in the formation of linear and cyclic oligonucleotides containing up to pentamers. Effects of temperature, time and surfactants have been examined. Rate constants for the formation of oligomers have been determined at five different temperatures. These data afforded values of H = 11.8 ± 1.9 Kcal mole^–1, S=–53 6 e.u. and G = 27.4 4.0 Kcal mole^–1. Prebiotic significance of these results are discussed.     

Porosity and available water of temporarily waterlogged soils in a Quercus robur (L.) declining stand

Pedunculate oak (Quercus robur L.) is particularly sensitive to decline in clayey soils presenting a high-perched temporary water table. These soils induce two successive constraints in one-year cycle: water excess (and hypoxy) in winter and early spring, and water shortage in summer (water stress being more restrictive to oak). We determined the porosity and water properties of temporarily waterlogged clayey soils supporting forest stands of decliningQuercus robur trees in a 101yr-old oak stand in Belgium (50°06N, 4°16E). Roots unevenly colonized the soil down to 1.6m: fine roots (diameter<5mm) were mostly distributed on the surface horizons (0–0.3 m) and around 1.3m deep, despite dense clayey horizons appearing at 0.35m depth. Clay content below this depth reached 46–51. Illite and vermiculite were the dominant clay minerals. The clayey horizons exhibited marked shrink–swell properties: bulk density at 30kPa increased from 1.41 to 1.88gcm^–3 from the surface to 2m depth. There was also evidence of hypoxic conditions caused by water saturation of pore space in the rooting zone from October to mid-April. Extractable water (EW), calculated between moisture tensions of 5 and 1600kPa was 152.8mm. The level of perched temporary water table strongly depended on the seasonal rhythm of water uptake by trees and on the shrink–swell behaviour of soil.     

Soybean β-Conglycinin α Subunit is Phosphorylated on Two Distinct Serines by Protein Kinase CK2 In Vitro

Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean -conglycinin subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. -Conglycinin subunit (68 kDa) presents seven consensus sequences, but only 0.5–1 mol P/mol subunit was incorporated by CK2. [³²P]Phosphorylated -conglycinin subunit was cleaved either by cyanogen bromide or by trypsin. ³²P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70–89, 116–126, and 116–127 sequences. Over the seven consensus sequences of -conglycinin subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites.     

INFLUENCE DU SUPERPARASITISME SUR LA DURÉE DU DÉVELOPPEMENT LARVAIRE ET LE POIDS DU PARASITOÏDE LIXOPHAGA DIATRAEAEÉLEVÉ DANS UN HÔTE DE SUBSTITUTION GALLERIA MELLONELLA

Résumé G. mellonella infestée au dernier stade larvaire avec 1, 2, 3 ou 5 planidia/hôte (ph/H) produit 1 à 5 pupes/hôte (pu/H). La mortalité des chenilles augmente avec le nombre de pl/H. Le poids des pupes et décroît avec un nombre croissant de pu/H (18,2 à 12,9 mg pour les et 12,5 à 9,7 mg pour les ). Le développement larvaire dure 8,7 j. chez les et 8,3 chez les ; il est peu affecté par le superparasitisme. Avec 1, 2, 3 et 5 pl/H nous obtenons 0,84–1,61–2,17 et 3,43 pu/H et 0,81–1,48–2,10 et 3,11 imagos/H. L'optimum est de 3 pl/H ou 1 à 2=pl/H pour obtenir des parasitoïdes plus lourds.

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Summary The influence of superparasitism on larval and pupal development is investigated. Last-larval instar G. mellonella (200±5 mg) were infected with 1, 2, 3 or 5 planidia/host (pl/H) producing 1 to 5 pupae per host (pu/H). Host mortality (8.6–8.3–14.3 and 22.2%) increased with the number of planidia. The planidia may transmit a bacteriosis. Pupal weight ( and ) decreased as number of pu/H increased. pupae were heavier than ones: 12.9 to 18.2 mg for against 9.7 to 12.5 mg for . Larval development lasted slightly longer for (8.7 d.) than for (8.3 d.), and its duration was little affected by superparasitism. 1, 2, 3 or 5 pl/H yielded 0.84–1.61–2.17 and 3.43 pu/H and 0.81–1.48–2.10 and 3.11 adults/H. An optimum was obtained with 3 planidia of L. diatraeae on G. mellonella or 1 to 2 to obtain heavier parasitoids.

     

Seasonal variation of carrageenans in tetrasporic,cystocarpic and ‘sterile’ stages of Gigartina skottsbergii S. et G. (Rhodophyta,Gigartinales)

Cystocarpic and sterile plants of Gigartina skottsbergii produce -carrageenans, while tetrasporophytes produce -carrageenans. No seasonal variation in the carrageenan yields in the three stages was evident. Yields were clearly higher in cystocarpic (34.1–64.7%) and sterile samples (32.3–71.8%) than in tetrasporic plants (10.1–34.0%). Chemical characteristics of the polysaccharides for any stage are the same throughout the year.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Continuous production of α,α-trehalose by immobilised fungal trehalose phosphorylase

Immobilisation of trehalose phosphorylase from Schizophyllum commune by adsorption onto anion-exchange materials stabilised the enzyme activity at 30°C by approx. 35-fold. Immobilised and free enzymes showed similar pH-dependence of activity but different inactivation behavior above 30°C. A fixed-bed enzyme reactor produced ,-trehalose at a stable substrate conversion of 80% with a productivity of 2.6 g l–1 h–1 for 72 h. Inhibition of trehalose phosphorylase by phosphate limited the productivity of a direct conversion of starch into ,-trehalose.     

An efficient cryopreservation procedure for potato (Solanum tuberosum L.) utilizing the new ice blocking agent, Supercool X1000

Cryopreservation has been recognized as a practical and efficient tool for long-term storage of vegetatively propagated plants. This study was conducted to investigate effects of modified vitrification techniques on cryopreservation of potato. In vitro plants of potato cultivars Superior and Atlantic were cold acclimated, and axillary buds were precultured, osmoprotected, exposed to PVS-2 solution, plunged into liquid nitrogen, thawed, and finally planted in the regeneration medium. In the modified vitrification technique an ice-blocking agent, Supercool X1000, was added with PVS-2 solution. Cold acclimation affected survival of cryopreserved shoot tips, and the highest survival (46.7%) was obtained after 3 weeks of acclimation at 10°C. Shoot tips exposed to 2M glycerol plus 0.6M sucrose for 40 min gave 51.5% and 11.7% survival in Atlantic and Superior at 10°C, respectively. Cold acclimated and osmoprotected shoot tips were dehydrated with PVS-2 containing different concentrations of Supercool X1000 prior to a plunge into liquid nitrogen. Treatments with 0.1% and 1% of Supercool X1000 significantly improved survival by 55% in Superior and 71.3% in Atlantic, respectively. After cryopreservation, vitrified shoot tips resumed growth within a week in a medium (1 mg l^–1 GA₃, 0.5 mg l^–1 zeatin, and 0.1 mg l^–1 IAA) with a low level of Pluronic F-68 (0.005%) and survival was 33.7% higher in Atlantic and 14.7% higher in Superior than the control (without Pluronic F-68).     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

Effects of specific inhibitors of cellular functions on sulfur mustard-induced cell death

This study was conducted to determine whether inhibitors of normal cellular functions can reduce cytotoxicity induced by sulfur mustard (HD). The compounds examined include inhibitors ofpoly(ADP-ribose) polymerase (PADPRP), inhibitors of mono(ADP-ribose) transferase (MADPRT), inhibitors of lipidperoxidation, and an inhibitor ofprotein synthesis. To determine the effects of these compounds on HD-induced cell death, human lymphocyte preparations were treated with known concentrations (0.1 M to 1000 M) of an inhibitor and exposed to an estimated 87% effect concentration (EC₈₇) of HD (170 M) for loss in cell viability. Cell viability was determined at 24–26hr post-exposure to HD using a dye (propidium iodide) exclusion assay and a flow cytometer. All of the selected PADPRP inhibitors were found to be effective at reducing the cytotoxic effects of HD. These inhibitors were rank-ordered based on the concentration that gives 50% (EC₅₀) reduction ofHD-induced cell death.A signijicant correlation (r=0.94) was observed between the compounds' ability to inhibit PADPRP and the compounds' ability to reduce HD- induced cell death, suggesting that PADPRP plays a role in HD-induced cell death. Inhibitors of MADPRT, lipid peroxidation, and protein synthesis were not effective at reducing HD-induced cell death.Abbreviations ATP adenosine triphosphate - DNA deoxyribonucleic acid - EC₅₀ concentration which gives 50% of maximum effect - GSH glutathione - HD sulfur mustard (,-dichloroethyl sulfide) - HEPA high efficiency particulate adsorbing - HEGA high efficiency gas adsorbing - IC₅₀ concentration that inhibits 50% of enzyme activity - MADPRT mono(ADP-ribose) transferase - NAD nicotinamide adenine dinucleotide - PADPRP poly(ADP-ribose) polymerase     

Stable carbon isotope signatures of background tropospheric chloromethane and CFC113

Samples of background air were collected in thelower troposphere of the Northern (high Arctic,northern Ontario, Vancouver and Houston) andSouthern (Baring Head, New Zealand) Hemispheresover the period July 1999 until March 2001.These samples were analysed for the stablecarbon isotope ratios of1,1,1-trichlorotrifluoroethane (CFC113) andCH₃Cl using a gaschromatography-continuous flow on-linecombustion isotope ratio mass spectrometrycombination. For CH₃Cl the global averageof the stable carbon isotope ratio is –36.2± 0.3 (error of mean). The average isbased on 78 data points, standard deviation forall measurements is 2.3, and the 90%confidence interval is –35.8 to –36.6.However, the number of data points from theSouthern Hemisphere is rather limited and thusthis observation is not necessarilyrepresentative for the entire SouthernHemisphere. A simple isotopic budget ofCH₃Cl shows the most important parametersneeding to be defined are the kinetic isotopeeffect of CH₃Cl destruction by OH radicalsand the source composition of CH₃Clemitted by the oceans and biomass burning ofC-4 plants. Present budgets of atmosphericCH₃Cl show a significant deficit in thesource strength. We estimate that the averagestable carbon isotope ratio of the additionalCH₃Cl emissions required to balance thebudget is –41.9 ± 7.8. The averageCFC113 isotopic composition based on 38measurements is –23.3 ± 1.6 (error ofmean), = 9.6 with no significantdifference between the hemispheres.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Avoidance Responses to Infrasound in Downstream Migrating European Silver Eels, Anguilla anguilla

In an attempt to develop an efficient acoustic fish fence, we have designed an infrasound source able to generate large nearfield particle acceleration. The source generates water movements by means of two symmetrical pistons in an air-filled cylinder with 21cm bore. The pistons are driven by eccentric coupling to an electric motor, with 5cm p.p. amplitude. The piston movements are 180° out of phase. The piston reaction forces are thus opposed, leading to vibration free operation. The submergible infrasound source is operated freely suspended in the water mass. The emitted sound frequency is 11.8Hz. The particle acceleration is about 0.01ms^–2 at a distance of 3m, corresponding to the threshold intensity for deterring effects of infrasound on Atlantic salmon smolts. The sound source was employed to test the effect of intense infrasound on migrating European silver eels. Fish confined in a tank displayed startle behaviour and prolonged stress reactions, telemetrically monitored as tachycardia, in response to intense infrasound. The field tests were carried out in the River Imsa. A trap that catches all the descending eels is installed near the river mouth. The trap was separated in four equal sections. During the periods with infrasound exposure, the proportion of silver eels entering the section closest to the sound source was reduced to 43% of the control value. In the section closest to the opposite river bank, infrasound increased the proportion of trapped eels to 144% of the control values. This shift of the migrating eels away from the infrasound source was highly significant.     

Contribution to the Biology of the Vermilion Snapper, Rhomboplites aurorubens, in Trinidad and Tobago, West Indies

Reproduction and growth of the vermilion snapper, Rhomboplites aurorubens, were studied in Trinidad and Tobago. The smallest individual caught measured 145mm total length (TL) and all fish appeared to be mature. It was not possible to precisely determine size at first maturity due to the use of macroscopic techniques. The smallest spent male and female measured 181 and 211mm TL respectively, suggesting a size at first maturity below these sizes. Spawning occurred throughout the year, with a period of peak spawning from about June to November in the rainy season when river runoff increased. Sagittal otolith sections were used for age determination and the opaque ring, which was counted as the annulus, was deposited from January to May in the dry season. A total of 11 age groups between the ages of 2–12 years (155–505mm total length) were found. The von Bertalanffy growth parameters were: L=532mm, K=0.13y^–1, and t₀=–0.17, where L is the asymptotic length, K is the growth coefficient and t₀ is the theoretical age at zero length. The relationship between weight (WT) and length (TL) was WT=3.43×10^–5 TL^2.82. Vermilion snapper in this study area appears to grow slower and attain a smaller asymptotic length, but has a longer lifespan than found in populations in higher latitudes. This may be attributed to different levels of exploitation, which may be higher in the latter areas.     

Two gerbils of the Negev: A long-term investigation of optimal habitat selection and its consequences

Optimal foraging theory has entered a new phase. It is not so much tested as used. It helps behavioural ecologists discover the nature of the information in an animals brain. It helps population ecologists reveal coefficients of interaction and their patterns of density-dependent variation. And it helps community ecologists examine niche relationships. In our studies on two species of Negev desert gerbil, we have taken advantage of the second and third of these functions. Both these gerbils prefer semi-stabilized dune habitat, and both altered their selective use of this habitat and stabilized sand according to experimental changes we made in their populations. Their changes in selectivity agree with a type of optimal foraging theory called isoleg theory. Isoleg theories provide examples of dipswitch theories – bundles of articulated qualitative predictions – that are easier to falsify than single qualitative predictions. By linking behaviour to population dynamics through isoleg theory, we were able to use the behaviour of the gerbils to reveal the shapes of their competitive isoclines. These have the peculiar non-linear shapes predicted by optimal foraging theory. Finally, when owl predation threatens, the behaviour of Gerbillus allenbyi reveals the shape of their victim isocline. As has long been predicted by predation theory and laboratory experiments, it is unimodal.     

Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy