Effects of Organic Nutrients and Hormones on Growth and Development of Tissue Explants from Coconut (Cocos nucifera) and Date (Phoenix dactylifera) Palms Cultured in vitro

The growth response (increase in weight) of cultured explants from seedling date (Phoenix dactylifera L.) and mature coconut (Cocos nucifera L. cv. Malayan Dwarf) palms to source and concentration of organic nitrogen. carbohydrate, auxins, cytokinins and gibberellins was examined. Growth was strongly stimulated by the presence of auxins (10^?7 to 10^?6M), cytokinins (10^?6 to 10^?5M), high concentrations of sucrose (0.2 M), and in the absence of NH₄Cl, by organic sources of reduced nitrogen. Higher concentrations of auxin (2,4-D or NAA at 10^?6 to 10^?5M) which still stimulated growth of Phoenix tissue, proved inhibitory to growth of freshly excised Cocos tissues. Explants from both palms initiated roots when subcultured on a medium with increased levels of auxin (NAA, 2.5 × 10^?6 to 2.5 × 10^?5M) and reduced levels of cytokinin (6-BAP, 5 × 10^?8M). Isolated roots excised from these explants continued growth and produced new laterals when subcultured on media with GA₃ (5 × 10^?7M) and reduced levels of auxin, cytokinin, and either minerals or sucrose.     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: GABA- and other transmitter receptors

Summary Transmitter receptor ion channels from previously identified rabbit olfactory bulb neurons were studied by using a thin slice preparation in combination with patch-clamp measurements. PG cells, which closely resembled previously described periglomerular interneurons in their morphology, responded to microapplication of GABA, acetylcholine, norepinephrine and glycine with the activation of distinct ionic currents. JG cells, which belong either to the class of short axon cells or external tufted cells, never showed GABA responses. In mitral cells ionic currents activated by GABA, acetylcholine, norepinephrine and glutamate could be elicited. Further measurements of GABA-activated currents of PG cells were made and indicated that these cells expressed two different types of GABA receptors: one which showed fast desensitization with a decay time constant of about 5 s, and one which slowly desensitized with a decay time constant of about 20–30 s. Both types were completely inhibited by bicuculline methiodide (50 M). GABA receptors were not blocked by Zn²⁺ (0.1 mM). From the dose-response relationship of the peak GABA-activated currents, an apparent dissociation constant of 50 M was derived. From single channel measurements in excised outside-out patches, a single channel conductance of GABA-activated Cl^– currents of 24 pS was obtained during continuous application of the agonist. Single channel events had a mean open time of 1.9 ms.     

Excitatory neuromuscular transmission in crayfish: Calcium dependence is unaffected by picrotoxin

Picrotoxin, 1 × 10^?5M to 1.6 × 10^?3M, had little or no effect on the amplitude of intracellularly recorded excitatory junctional potentials (EJPs) at extracellular calcium concentrations [Ca²⁺]₀ ranging from 0.5 to 15 mM. The slope of the log EJP vs. log[Ca²⁺]₀ relationship was approximately 1 with or without picrotoxin. The reduction of EJP amplitude resulting from the addition of 5 × 10^?5M GABA was largely reversed by 10^?5M picrotoxin.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

In vitro Plant Formation from Stem Explants of Rape (Brassica napus cv. Zephyr)

Internodal segments from 6-weeks-old rape plants (Brassica napus L. cv. Zephyr) were induced to differentiate in vitro producing shoots or shoots and roots on synthetic nutrient medium under controlled conditions. Benzyladenine (BA) alone (5 × 10^?6 M) induced multiple shoot formation on all stem explants. Roots were induced on shoots when recultured on nutrient medium supplemented with auxins such as naphthalene-acetic acid (NAA) or indoleacetic acid (1AA) or when planted in vermiculite. Complete plant formation was obtained when NAA (2 × 1^?6, 5 × 10^?6 and 10^?5 M) was employed in conjunction with BA at 5 × 10^?6M. At higher concentrations (10^?5M) NAA retards the shoot development while 1AA suppresses it totally. Lower levels of auxins along with the cytokinin did not retard or inhibit shoot differentiation.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Stimulation of Growth of Peppermint (Mentha piperita) by Phosfon, a Growth Retardant

The shoot growth and fresh weight of Mentha piperita grown in soil were stimulated at concentrations of 1.26 × 10^?5M to 7.77 × 10^?4M phosfon (2,4-dichlorobenzyl tributyl phosphonium chloride) while higher concentrations resulted in retardation of growth. Concentrations of 6.30 × 10^?7M to 3.78 × 10^?5M caused retardation of growth in mineral nutrient solution, and even death at the highest concentrations. However, when the M. piperita plants were grown in mineral nutrient solutions at concentrations of phosfon which had been sequentially lowered from 2.52 × 10^?8M to 2.52 × 10^?12M, the shoot growth and fresh weight were stimulated as in the case of plants grown in phosfon treated soil.     

Growth and Geotropic Responses of Avena Coleoptiles to 4-Amino-3,5,6-Trichloropicolinic Acid

The elongation and geotropic responses of coleoptile sections of Avena sativa L. to various concentrations of 4-amino-3,5,6-trichloropicolinic acid (Tordon) proved to be qualitatively similar to those previously reported for 2,3,6-trichlorobenzoic acid (TCBA). Tordon stimulated growth in a range of concentrations from 1 × 10^?6 to 1 × 10^?4M but higher concentrations were inhibitory. Geotropic curvature was extensively depressed by 1 × 10^?5 and 1 × 10^?4M Tordon, concentrations which accelerated elongation. A similar differential effect has been reported for TCBA and other auxins. Several other picolinic acids and related compounds were tested, but only very slight responses were noted.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

GABA inactivation at the crayfish neuromuscular junction

Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10^?5 to 5 × 10^?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.     

The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

As a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil-grown plants and in vitro-grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10^?6 M-1 ×10^?4 M) of the auxins 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and mdole-3-acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil-grown plants. The cytokinins examined 6-benzyl-aminopurine (BAP) and 6-4-hydroxy-3-methyl-2-enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10^?6 M in combination with BAP at a concentration of 1× 10^?6 or 1 × 10^?4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10^?6 M) combined with BAP (1 × 10^?6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil-grown plants, and NAA (1 × 10^?5 M) combined with BAP (1 × 10^?6 or 1 × 10^?5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4-D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4-D and BAP at concentrations of 1 × 10^?6 M allowed differentiation of the resistant and susceptible cultivars. These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.     

Promoting Effect of Fusicoccin on Seed Germination

The effects of fusicoccin on the germination of dormant, light-requiring or abscisic acid-inhibited seeds has been investigated. (1) Fusicoccin (10^?6M) induces germination in dormant wheat seeds (Triticum durum cv. Cappelli; 1972 crop) and stimulates it in seeds already relieved from dormancy (1971 crop), with an effect similar to that of gibberellic acid. (2) Fusicoccin (1.5 × 10^?6M) is more active than the two phytohormones gibberellic acid and benzyladenine and than white light in stimulating light-requiring lettuce seeds (Lactuca sativa cv. Grand Rapids) to germinate. Germination of radish seeds (Raphanus sativus) is also accelerated by fusicoccin, while benzyladenine and gibberellic acid are less active in this material. (3) Fusicoccin (1.5 × 10^?5M) removes almost completely the inhibitory effect of abscisic acid on germination of radish and lettuce seeds, whereas benzyladenine (10^?4M) and gibberellic acid (3 × 10^?4M) remove the inhibition only partially. The possible relationship between these results and previous information on growth by cell enlargement is discussed in terms of the mechanism of action of fusicoccin as compared with natural hormones.     

Effect of ortho-dihydroxyphenols on growth and protein pattern of callus cultures from Prunus avium

Excised shoot discs from Prunus avium L. were cultivated on a modified Murashige and Skoog medium for 4 weeks under long day conditions. The basal medium was supplemented with IAA and benzylaminopurine. Further addition of 5.7 × 10^?5 resp, 11.4 × 10^?5M catechin and 4.25 × 10^?5 resp. 8.5 × 10^?5M chlorogenic acid resulted in a highly significant promotion of callus proliferation. Proteins and enzymes were separated by isoelectric focusing. The supply of both polyphenols resulted in a higher number and intensity of protein bands including esterases as compared to controls.     

Properties of glutamate dehydrogenase from Beta vulgaris

Glutamate-NAD oxidoreductase, E.C. 1.4.1.3 (GDH), from seedlings of Beta vulgaris cv. Rota, Jahnsch Peragis Comp., was enzymatically characterized. This enzyme with molecular weight of 2.6 × 10⁵ has a pH optimum of around 8 for animation of α-KGA and around 9.5 for the desamination of glutamate. The apparent K_m for α-KGA is 6.7 × 10^?4M, for NH₃ 2.5 × 10^?3M, for NADH 3.2 × 10^?5M and for NAADPH 5.5 × 10^?4M. NAD¹ inhibits the reaction non-competitively when NADPH serves as substrate. The apparent K¹ is 4.5 × 10^?4M. The data are discussed on relation to the properties of GDH from other plant sources.     

Titration of Photophosphorylation, Oxidative Phosphorylation and Glycolysis in Scenedesmus with Desaspidin: Regulation between Pathways and Sites for Photophosphorylation

Narrow concentration intervals were used, covering 10^?6– 10^?4M desaspidin. The interaction with glycolysis involves three steps, the inhibitor constants (K_i:s) being in turn 2.7 × 10^?5M, 1.3 × 10^?4M, and high. About 18% of total glycolysis is inhibited in each of the two first steps, and 65% left for the third reaction. After compensation for glycolysis, oxidative phosphorylation may show a sudden jump to about 10% inhibition at 1.5 × 10^?5M desaspidin, the possible K_i of the reaction starting here being very high. Correcting for glycolysis, desaspidin affects total Photophosphorylation in two steps, with the K_i values of 7.8 × 10^?5M and 4.6 × 10^?4M respectively. Inhibition in the first step is about 27% of the total photophosphorylation. By applying 10^?6M DCMU[/3-(3, 4-dichlorophenyl)-l, l-dimethy lurea], one can abolish non-cyclic photophosphorylation. Desaspidin then reacts in a single step with a K_i of 1.4 × 10^?4M. At 5 × 10^?5M DCMU, also the pseudocyclic photophosphorylation is abolished. The remaining, true cyclic photophosphorylation has a single K_i of 2.3 × 10^?5M for desaspidin. Under non-cyclic conditions, the true cyclic process contributes about 25% to total Photophosphorylation. Under pseudocyclic conditions, no cyclic photophosphorylation occurs. Under true cyclic conditions, the non-cyclic and pseudocyclic processes are inoperative. This indicates a regulative system, so that either (1) the (non-cyclic + true cyclic), (2) only the pseudocyclic, or (3) only the true cyclic systems can be traced, dependent on the level of DCMU applied. There are two sites for non-cyclic Photophosphorylation, one of them common to the pseudocyclic pathway. Cyclic photophosphorylation has a third site, different from the other two.     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.     

Spontaneous bioelectric activity of cultured purkinje cells during exposure to glutamate,glycine, and strychnine

The addition of glutamate to the bathing medium increased the average firing rate of cerebellar rat Purkinje cells in vitro. At concentrations lower than 10^?6 M, there was no deviation from controls in the firing pattern or rate that was detectable. At 10^?3 M glutamate, the amplitude of the action potentials was gradually decreased until all activity was abolished. The action of glutamate was rapid in onset and reversible. Glycine produced sustained depression of firing at concentrations higher than 10^?3 M. This inhibition was strychnine-insensitive and considered nonspecific. Strychnine, on the other hand, exerted an excitatory influence on Purkinje cells when applied at low concentrations (10^?8 to 10^?6 M). The firing became more irregular and complex discharges appeared. Higher concentrations of strychnine (>10^?5 M) inhibited the spontaneous activity. The effect of strychnine was partly reversible. The data suggest that low concentrations of strychnine lower the threshold for inputs at excitatory as well as inhibitory synapses.     

A protocol for in vitro mass propagation of asiatic hybrids of lily through liquid stationary culture

Summary A simple, rapid and cost-effective in vitro scheme has been proposed for mass propagating two cultivars of Asiatic lily hybrids. An average of seven bulblets was formed after 17 d when 1×1 cm² bulb scale segments (explants) were cultured on Murashige and Skoog (MS) medium with 3% sucrose and 0.5 μM α-naphthaleneacetic acid (NAA). On MS medium containing 0.5 μM NAA and 6 or 9% sucrose, depending on the cultivar, large numbers of bulblets of increased size (3.5–5.0 cm in circumference) were formed under a 16/8 h photoperiod. A continuous system of mass propagation of bulblets was achieved through in vitro scale formation (secondary explants) on MS medium supplemented with 23 μM kinetin and 0.5 μM NAA, as well as scale proliferation on MS basal liquid stationary medium. Upon transplantation all bulblets sprouted, of which 40% flowered in the first season. Under ideal conditions, ca. 9.68×10⁵ bulblets can be produced from a single scale segment in 1 yr by following the systematic propagation steps proposed here.     

Restorative effects of cyclic AMP on complex bioelectric activities of cultured fetal rodent CNS tissues after acute CA++-deprivation

Explants of fetal mouse spinal cord and cerebral cortex generate organotypic slow-wave and repetitive-spike discharges in vitro which can be abolished by agents which reduce the concentration of Ca⁺⁺ available to the tissue. Synaptically mediated discharges are rapidly blocked in Ca⁺⁺-free balanced salt solution (BSS), or in regular BSS after addition of 10^?3 M EGTA, 5–10 × 10^?3 M Mg⁺⁺, or 10^?4 M xylocaine, but simple spike potentials can still propagate. When low concentrations of cyclic AMP or dibutyryl cyclic AMP (2 × 10^?6 M) are added to the Ca ⁺⁺-free BSS or Ca⁺⁺-antagonist-BSS, a temporary (1–20 min) restoration of characteristic complex bioelectric activities occurs (or the onset of depression is delayed if cyclic AMP is initially added). Phosphodiesterase inhibitors, e.g. 10^?3 M caffeine, are also effective in restoring these blockades, whereas 5′AMP and ATP are not. Application of 10^?6 M cyclic AMP or 10^?3 M caffeine in regular BSS greatly enhances excitability of some CNS explants, resembling convulsive effects observed in CNS in situ. The data suggest that cyclic AMP can mobilize Ca⁺⁺ from membranebound Ca pools within neurons in CNS explants so as to permit Ca⁺⁺-dependent release of neurotransmitter during Ca⁺⁺ deficits. Thus, it may also be that under normal conditions, cyclic AMP can regulate the availability of Ca⁺⁺ for synaptic transmission in the central nervous system, thereby modulating the efficacy of synaptic functions.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.