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     

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

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

Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator

We investigated retinol effects in ornithine decarboxylase activity in Sertoli cells. We also tested the hypothesis that free radical scavengers and iron chelators may attenuate the effect of retinol. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with retinol by 24 h with or without mannitol (1 mM) or 1,10 phenanthroline (100 M). We measured ornithine decarboxylase and catalase activities and malondialdehyde concentrations in response to retinol treatment. In response to 7 M retinol treatment ornithine decarboxylase activity increased 30%. Retinol-induced ornithine decarboxylase activity was significantly decreased by addition of free radical scavenger (mannitol) or iron chelator (1,10 phenanthroline). In addition the same effect was observed in catalase increased activity and in malondialdehyde concentrations. These results suggest that retinol treatment induced ornithine decarboxylase and catalase activity and increased malondialdehyde concentration. These effects appear to be mediate by ROS.     

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Optimization of in vivo tumor targeting in SCID mice with divalent forms of 741F8 anti-c-erbB-2 single-chain Fv: effects of dose escalation and repeated i.v. administration

Single-chain Fv molecules in monovalent (sFv) and divalent [(sFv')₂] forms exhibit highly specific tumor targeting in mice as a result of their small size and rapid systemic clearance. As a consequence, there is a rapid reversal of the sFv blood/tumor gradient, resulting in diminished retention of sFv species in tumors. In this report we investigate two distinct strategies, dose escalation and repetitive intravenous (i.v.) dosing, aiming to increase the absolute selective retention of radiolabeled anti-c-erbB-2¹²⁵I-741F8 (sFv')₂ in c-erbB-2-overexpressing SK-OV-3 tumors in mice with severe combined immunodeficiency (SCID). A doseescalation strategy was applied to single i.v. injections of¹²⁵I-741F8 (sFv')₂. Doses from 50 g to 1000 g were administered without a significant decrease in tumor targeting or specificity. High doses resulted in large increases in the absolute retention of¹²⁵I-741F8 (sFv')₂. For example, raising the administered dose from 50 g to 1000 g increased the tumor retention 24 h after injection from 0.46 g/g to 9.5 g/g, and resulted in a net increase of greater than 9 /g. Over the same dose range, the liver retention rose from 0.06 g/g to 1 g/g, and resulted in a net increase of less than 1 g/g. The retention of 9.5 g/g in tumor 24 h fllowing the 1000-g dose of (sFv')₂ was comparable to that seen 24 h after a 50-g dose of¹²⁵I-741F8 IgG, indicating that the use of large doses of (sFv')₂ may partially offset their rapid clearance. When two doses were administered by i.v. injection 24 h apart, the specificity of delivery to tumor observed after the first dose was maintained following the second injection. Tumor retention of¹²⁵I-741F8 (sFv')₂ was 0.32 g/g at 24 h and 0.22 g/g at 48 h following a single injection of 20 g/g, while 0.04 g/ml and 0.03 g/ml were retained in blood at the same assay times. After a second 20-g injection at the 24-h assay time, tumor retention increased to 0.49 g/g, and blood retention was 0.06 g/ml, at the 48-h point. These results suggest that multiple high-dose administrations of radiolabeled 741F8 (sFv')₂ may lead to the selective tumor localization of therapeutic radiation doses.Supported by National Cancer Institute (NCI) National Cooperative Drug Discovery Group grant U01 CA51880, CA06927, an appropriation from the Commonwealth of Pennsylvania, and the Bernard A. and Rebecca S. Bernard Foundation     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Antihuman immunodeficiency virus (HIV-1) activities of inhibitors of polyamine pathways

Four inhibitors of polyamine biosynthetic pathways were tested for their effect on HIV-1 replication in phytohemagglutinin-stimulated human peripheral blood mononuclear cells. Methyl acetylenic putrescine (MAP) and -monofluoromethyldehydroornithine methyl ester, irreversible inhibitors of ornithine decarboxylase, inhibited the production of p24 antigen in phytohemagglutinin-stimulated peripheral blood mononuclear cells by clinical HIV-1 strains isolated from HIV-infected patients with IC₅₀ values of about 1–2 µM. 5-{(Z)-4-amino-2-butenyl]methylamino}-5-deoxyadenosine (MDL 73811), an enzyme-activated irreversible inhibitor of S-adenosyl-L-methionine (AdoMet) decarboxylase, also inhibited the production of p24 antigen by HIV-1 strains in peripheral blood mononuclear cells with IC₅₀ values of 1–2 µM. The least potent was 1-aminoxyethylamine which is another inhibitor of AdoMet decarboxylase. MAP showed the best therapeutic index of 500–1,000.     

Choline Administration Reverses Hypotension In Spinal Cord Transected Rats: The Involvement of Vasopressin

Intracerebroventricular (i.c.v.) choline (50–150 g) increased blood pressure and decreased heart rate in spinal cord transected, hypotensive rats. Choline administered intraperitoneally (60 mg/kg), also, increased blood pressure, but to a lesser extent. The pressor response to i.c.v. choline was associated with an increase in plasma vasopressin. Mecamylamine pretreatment (50 g; i.c.v.) blocked the pressor, bradycardic and vasopressin responses to choline (150 g). Atropine pretreatment (10 g; i.c.v.) abolished the bradycardia but failed to alter pressor and vasopressin responses. Hemicholinium-3 [HC-3 (20 g; i.c.v.)] pretreatment attenuated both bradycardia and pressor responses to choline. The vasopressin V1 receptor antagonist, (-mercapto-, -cyclopenta-methylenepropionyl¹, O-Me-Tyr², Arg⁸)-vasopressin (10 g/kg) administered intravenously 5 min after choline abolished the pressor response and attenuated the bradycardia-induced by choline. These data show that choline restores hypotension effectively by activating central nicotinic receptors via presynaptic mechanisms, in spinal shock. Choline-induced bradycardia is mediated by central nicotinic and muscarinic receptors. Increase in plasma vasopressin is involved in cardiovascular effects of choline.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Polyamines and related enzymes in rice seeds differing in germination potential

In ungerminated rice seeds, (Japonica rice variety, CV Tapei 309), the content of free amines (putrescine, spermidine, spermine, tyramine) was higher in seed lots having a low germination frequency compared to those with high germination potential. Conversely, amine conjugates (di-feruloylputrescine, di-feruloylspermidine, diferuloyldiaminopropane and feruloyltyramine) decreased with loss of viability. Thus, these compounds appeared to constitute biochemical markers of seed viability. In seeds with high germination potential, conjugates decreased drastically during germination, with an early and rapid increase in free amines (putrescine, spermidine, tyramine). Arginine decarboxylase (ADC) activity was highest during the germination of high germination potential seeds, its activity gradually declining with loss of viability and being closely correlated with agmatine content. The polyamine biosynthetic inhibitors (-DL-difluoromethylarginine, DFMA, a specific and irreversible inhibitor of ADC; -DL-difluoromethylornithine, DFMO, a specific irreversible inhibitor of ornithine decarboxylase (ODC); cyclohexylammonium sulfate, CHA, inhibitor of spermidine synthase) neither depleted putrescine and spermidine levels nor inhibited germination in high germination potential seeds. In low germination potential seeds, the germination process was inhibited by DFMA or CHA. Application of agmatine resulted in a reversal of inhibition. DFMA inhibited ADC activity in both categories of seeds. In low germination potential seeds treated with CHA no ADC activity was found. These results suggest that amines are involved in the germination process of rice seeds. It appears that amine conjugates may serve as a storage form of amines which, upon enzymatic hydrolysis, could supply the cell with an additional amine reserve and influence cell division and/or cell elongation.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

The gibberellin synthesis inhibitors,ancymidol and flurprimidol,promote in vitro rooting of white pine microshoots

Summary Ancymidol and flurprimidol were tested for their ability to induce in vitro rooting on axillary proliferated shoots of white pine (Pinus strobus L.). Shoots were treated for 30 days (pulse) with growth regulators, then subcultured to 0.5X medium for conifer morphogenesis without growth regulators. A pulse treatment containing 5 M ancymidol and 0.54 M naphthaleneacetic acid resulted in 43% rooted shoots, whereas a pulse treatment with 0.54 M naphthaleneacetic acid alone resulted in 7% root formation. Flurprimidol also stimulated rooting of white pine shoots, but was less effective than ancymidol. No detrimental effects on shoot growth were observed with the gibberellin synthesis inhibitors at the 5 M concentration used. Some rooted shoots were successfully acclimatized to the greenhouse.Abbreviations ancymidol -cyclopropyl-(4-methoxyphenyl)-5-pyrimidinemethanol - flurprimidol -(1-methylethyl--[4-trifluromethoxy)phenyl]-5-pyridinemethanol - GA gibberellin - MCM medium for conifer morphogenesis - NAA 1-naphthaleneacetic acid     

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.Abbreviations ADC arginine decarboxylase - Dap diaminopropane - DFMA -difluoromethylarginine - MGBG methylglyoxal-bis-guanylhydrazone - MTA 5-deoxy-5-methylthioadenosine - ODC ornithine decarboxylase - PA polyamines - PAO polyamine oxidase - PCA perchloric acid - PLP pyridoxal phosphate - Put putrescine - SAM S-adenosylmethionine - dSAM decarboxylated S-adenosylmethionine - SAMDC S-adenosylmethionine decarboxylase - Spd spermidine - Spm spermine     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.