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.     

In vitro allelopathic effects of extracts and amenthoflavone from Byrsonima crassa (Malpighiaceae)

Abstract

Extracts and pure amenthoflavone isolated from Byrsonima crassa (Malpighiaceae), a shrub growing in the semi-arid region of Brazil Cerrado, were evaluated in vitro, at different doses, for their effects on tomato seed germination and subsequent growth of seedlings. A hydromethanolic extract showed general stimulatory effects. The EtOAc extract stimulated root elongation and root weight of tomato; shoot elongation was inhibited, while shoot weight was not altered. The pure amenthoflavone isolated from the plant, stimulated shoot elongation at concentrations ranging between 10^?4 M and 10^?6 M.     

Non-Reversibility of Gibberellin-Induced Inhibition of Regeneration in Begonia Leaves

With applied to the petioles of detached Begonia x cheimantha leaves before planting, Gibberellic acid (GA₃) inhibited the formation of adventitious buds and roots ill an apparently irreversible manner. Bud formation was entirely suppressed by 10^?6M and higher concentrations and a significant inhibition was still present at 10^?9M the lowest concentration tested. Root formation was not affected by GA₃ below 10^?7M and was possible even at 10^?4 M GA₃. Petiole elongation was stimulated by GA₃ with an optimum at 10^?5M. GA₃ also blocked the action of 6-benzyiamino-purine (BAP) and 1-naphthaleneacetic acid (NAA), compounds which are potent stimulators of bud and root formation, respectively. When applied simultaneously with GA₃ they were, at their optimal concentrations, devoid of any effect in counteracting or reversing the gibberellin-induced inhibitions. Abscisic acid and the growth retardants CCC and Phosfon also were unable to restore bud and root formation. In leaves initially treated with water or 10^?5M BAP, endogenous bud and root formation as well as BAP-induced bud formation were entirety suppressed when 10^?5M GA₃ was applied 8 days after the initial treatments. Even when delayed for 14 days GA₃ treatment inhibited BAP-induced bud formation, while treatment after 21 days bad little effect on bud and root formation. Development of pre-existing, visible bud primordia was not inhibited by GA₃. BAP and NAA competitively inhibited the action of GA₃ in petiole extension growth. The results are discussed in relation to results obtained in other plant systems. It is suggested that GA₃ acts by blocking of the organized cell divisions initiating the formation of bud and root primordia.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

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.     

Is Cell Elongation Regulated by Extracellular Auxin?

Experiments with isolated epidermal strips of maize coleoptiles, pretreated with auxin and further incubated on sucrose agar containing different concentrations of auxin (indole-3-acetic acid, IAA or naphthalene-1-acetic acid, NAA) and/or naphthylphthalamic acid (NPA), are described. Preincubation for 2h with 2 . 10^?4M IAA or 10^?5M NAA in buffer, followed by 30 min wash in buffer results in measurable cell elongation during a subsequent incubation for 6 h on sucrose agar. Addition of 10^?4M NPA inhibited the response to auxin and this inhibition could be reversed by providing IAA in addition to NPA. Inner tissue fragments (without outer epidermis) did not respond to external IAA. These results lead to the conclusion that auxin secretion at the outer epidermis may be an essential step in auxin-regulated coleoptile growth.     

Effect of Gibberellic Acid on the Elongation Rate of Agrostis alba Root Hairs

The influence of gibberellic acid over a wide range of concentrations on the rate of elongation of root hairs of redtop grass was investigated. The rate of root hair elongation was increased by GA over the concentration range of 10^?7 to 10^?12 M inclusive, with peak stimulation occurring at 10^?6 M. Although root hair growth was slightly accelerated by 10^?6 M GA, this concentration damaged many root hairs and caused some to stop growing altogether. Rate of root hair elongation was reduced to less than 84% of the control by 10^?5 M GA.     

AXENIC TISSUE CULTURES FROM THE SPOROPHYTES OF LAMINARIA DIGITATA AND LAMINARIA HYPERBOREA (PHAEOPHYTA)

Starting material for the tissue cultures was the meristematic basal zone of the blade. Pieces treated 30–60 sec in hypochlorite solution were rinsed and placed on agar plates made from the artificial seawater ASP6 F2 solidified with 6 g agar l^?1. After 6 weeks colorless callus-like tissue grew out from some pieces. Treatment with activated charcoal removed some inhibiting substances from the agar medium as numbers of callus developing pieces increased on such plates. A combination of 10^?5 M NAA and 5 · 10^?7 M kinetin gave a yellow-brown tissue. A differentiation in the tissue from L. hyperborea was observed as well as the formation of meiospores, which grew out into male and female plants. Thalli of sporophytes were observed but they never reached a length of more than one mm before they died or changed to an irregular pattern of growth.     

Physiological effects of betalains upon higher plants

Red and yellow betalains isolated from red beetroots by means of gel filtration were strong inhibitors of indole-3-acetic acid oxidase; 50% inhibition was obtained at 5 × 10^?7 M and 3 × 10^?7 M respectively. Concentrations of 10^?4 M betanin had no effect upon ATP production in mitochondria. The red pigment relieved the inhibitory effects upon wheat root elongation caused by indole-3-acetic acid but not the inhibition caused by 2,4-dichlorophenoxyacetic acid.     

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca²⁺-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca²⁺ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10^?6–10^?5 M and Ca²⁺ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.     

Resistance system of Mycobacterium bovis B.C.B. to aminoglycoside- and peptide-antibiotics. Comparison of resistant phenotypes between Mycobacterium tuberculosis and Mycobacterium bovis

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10^?5-10^?6, 10^?5-10^?6, and 10^?6-10^?7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.     

INFLUENCES OF COLCHICINE, VINBLASTINE AND CYTOCHALASIN ON THE RELEASE OF 5-HYDROXYTRYPTAMINE FROM RAT BRAIN SYNAPTOSOMES

Rat brain synaptosomes preincubated with [³H]5-HT. were further incubated and the release of [³H]5-HT from the preparation was studied. The spontaneous release consisted of an initial rapid phase followed by slower release. Incubation with 60 mM-KCl increased the release while 60 mw-NaCl did not affect it. The effect of KG was abolished when NaCl was omitted from the medium. The potassium-induced release was Ca²⁺ -dependent while that induced by tyramine (10^?5-10^?4M) and the spontaneous release did not depend on Ca²⁺. Vinblastine (10^?5–2.5 X 10^?4 M) caused an increase in the spontaneous release and an decrease in the potassium-induced release, while it completely inhibited the release by tyramine at 2.5 X 10^?4 M. Colchicine (5 X 10^?5–10^?3M) and cytochalasin D (10^?5, 10^?4 M) failed to produce any change in the release. Cytochalasin B (10^?5, 10^?4M) increased the spontaneous release and decreased the potassium-induced release but it did not affect the release by tyramine. Colchicine (10^?3 M). vinblastine (10^?4 M) and cytochalasin B (10^?4 M) did not affect significantly Na⁺.K⁺-. Mg²- and Ca²⁺ -ATPase activities. These results suggest that none of microtubules. microfilaments and contractile protein participates in the mechanism of [³H]5-HT release from synaptosomes, in vitro.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

The effect of nitrogen on the movement of tracers down the stolon of Saxifraga sarmentosa,with some observations on the influence of light

Summary The movement of applied ¹³⁷Cs and of naturally-assimilated ¹⁴C down the long stolon of Saxifraga is strongly inhibited by confining a length of 10 to 30 cm of the stolon in an atmosphere of nitrogen. The inhibition is reversible, normal transport being restored after less then 4 h when the stolon is returned to air from 5 h in nitrogen. Callose formation does not seem to be involved. There is evidence that local darkness has a similar adverse effect on phloem transport.These findings are considered antagonistic to the pressure-flow hypothesis, but favourable to the active mass-flow theories.This work formed part of that submitted for the degree of Ph.D. of the University of London.     

A study of regression and budding in Perophora viridis

1. A method has been devised for studying the regression of the zooid of Perophora into a stolon and the subsequent differentiation of a new zooid from this stolon. 2. Circulatory cells of the stolon resulting from regression will aggregate into masses larger than the minimal size necessary for differentiation of a zooid, but fail to differentiate into a zooid. 3. The cells of a zooid after staining with neutral red appear in the stolon during regression and finally come to lie in the newly formed zooid. 4. During the cycle of adult zooid to stolon to newly formed zooid, there is no evidence for cell division from studies with tritiated thymidine. 5. It is concluded that under conditions of starvation, an adult zooid furnishes all the cells for the formation of a stolon and the subsequent zooids without cell division.     

Induction of metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis Lamarck, 1819 using neuroactive compounds,KCl, NH4Cl and organic solvents

Pediveliger larvae of Mytilus galloprovincialis were subjected to a series of bioassays to investigate the induction of metamorphosis using neuroactive compounds, K⁺, NH₄ ⁺ and organic solvents. Growth and survival of post-larvae obtained using ethanol and methanol were also observed. Epinephrine, phenylephrine, clonidine and metanephrine induced larval metamorphosis at 10^?6 to 10^?4 M in both 24-h and continuous exposure assays. In 24-h exposure assays, α-methyldopa at 5×10^?5 M and methoxyphenamine at 5×10^?5?10^?4 M induced 55?94% metamorphosis. Similarly, excess K⁺ at 3×10^?2 M induced 39% metamorphosis and NH₄ ⁺ at 1?5×10^?2 M induced 63–78% metamorphosis. The EC50s of seven organic solvents ranged from 0.04 to 0.82 M. Post-larvae that metamorphosed using ethanol and methanol survived as juveniles and grew at the same rate as those from microbial biofilm. Thus, the above compounds can be useful inducers of metamorphosis for antifouling studies using larvae and juveniles of M. galloprovincialis.     

Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation

Summary Sunflowers are known to respond to Fe deficiency (-Fe) with a typical root tip swelling and the formation of root hairs and transfer cells in the rhizodermis. The possible regulation of this process was examined by a comparative study of root morphology and cytology of intact seedlings (Helianthus annuus L. cv. Giganteus) under -Fe and hormonal treatment in nutrient solution. Longitudinal sections of -Fe roots showed root tip swelling is due to cessation of cell elongation and isodiarnetric volume increase of the cortical cells. Enhanced cell division in the pericycle leads to the formation of lateral root primordia in the swollen zone. Xylem vessel differentiation is markedly accelerated and accompanied by early differentiation of the casparian band in the endodermis. Exogenous application of IAA (10^–8-10^–7 M) via the nutrient solution to Fe sufficient plants causes symptoms which closely mimick the characteristics of Fe deficiency including root hair development. Moreover, rhizodermal cells produce peripheral protuberances reminiscent of -Fe transfer cells. Ethylene-releasing ethephon (10^–4M) also causes subapical swelling and root hair formation. However, wall protuberance development is less pronounced. ABA (10^–5 M) leads to similar root thickening and root hair formation but without any comparable transfer cell differentiation. From the striking similarities between -Fe and IAA treatment it is concluded that this hormone (possibly in cooperation with ethylene) is involved in the Fe stress response of sunflower roots. The importance of a continuous polar IAA transport for this process is discussed.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - Ethephone 2-chloro-ethylphosphonic acid - Fe(III)-EDTA ethylenediaminetetraacetic ferric-sodium salt - IAA indole-acetic acid - TIBA triiodobenzoic acid     

Phospholipase A2 from Trypanosoma congolense: Characterization and haematological properties

Phospholipase A₂ was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl-sn-phosphatidylcholine and 1,2,dioleoyl-sn-phosphatidylcholine with K_m values of 1·85 × 10^?3 M and 2·12 × 10^?3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol^?1. Inhibition studies with parahydroxymercuribenzoate and tri-butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.