DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA: THE COCONUT-MILK PROBLEM

In an attempt to define a medium and to investigate the effects of glycine, floral buds of Aquilegia were grown in culture on coconut milk and defined media containing either White's minerals or a new mineral formula, with and without glycine. Development obtained on the best defined medium was 66 to 76% of that achieved on the equivalent coconut-milk medium. The inability of buds to achieve development equal to that on the coconut-milk medium may be partially explained by the existence of a barrier to carpel formation and development. The effect of glycine on floral buds grown on a defined medium was dependent not only upon the minerals used but also on the stage of development of the bud. With White's minerals, glycine inhibited the development of buds in early and intermediate stages but was slightly promotive with buds in later stages. With the new minerals, glycine was slightly promotive with early and intermediate stages of development but was inhibitory with later stages. Further work is necessary before development on a defined medium will equal or exceed development on the coconut-milk medium.     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi

The concentrations and composition of free amino acids in phloem sap from two cultivars of oats and barley, both susceptible to the aphid Rhopalosiphum padi, were determined by means of high performance liquid chromatography. Sap was collected from excised aphid stylets at three developmental stages (seedlings, tillering plants and plants undergoing stem elongation) from plants given or not given fertiliser and grown outdoors. In connection, the growth of individual R. padi nymphs was estimated at the same phenological stages on plants grown in the greenhouse. The content of free amino acids was consistently higher in seedlings than in plants at the early tillering stage. Only in seedlings did the addition of fertiliser increase amino acid levels. Barley phloem sap contained more free amino acids than that of oats when fertiliser was added and at later developmental stages. Phloem sap of oats and barley showed similar patterns in their composition of free amino acids at the seedling stage, but as the plants grew older the patterns became increasingly different. Plants given fertiliser had higher amounts of dicarboxylic amino acids (glutamic and aspartic acid) than unfertilised plants. The concentrations of γ-amino butyric acid, glycine, histidine, and methionine were very low in all treatments. The relative growth rates of R. padi nymphs were low when amino acid content was low and vice versa. The results are discussed in relation to host plant suitability and plant resistance mechanisms.     

CONTROL OF IN VITRO SEPAL DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA (RANUNCULACEAE)

Excised floral buds of Aquilegia formosa Fisch. were grown on a coconut-milk medium containing the minerals and vitamins of Murashige and Skoog, sucrose, and kinetin. The plant growth regulators indoleacetic acid (IAA, 0.5 mg/liter) and gibberellic acid (GA, 2.0 mg/liter) were added singly and in combinations; both were omitted from the control medium. The addition of GA to the basal medium was required to support sepal development on flowers at all phases of development. The formation of stomatal complexes in the epidermis of the sepals occurred only in the presence of GA. Sepals grown in the presence of GA also contained trichoblasts and developing trichomes, while none were formed in the absence of GA. The role of IAA in the development of these idioblasts was not clear but it appeared to have no effect. The hormones GA and IAA had different effects on the growth of the sepals. In the presence of GA the sepals increase in length until comparable with sepals grown in vivo. However, the sepals remained small when GA was omitted from the medium. Upon closer examination of this effect, it was determined that there was a direct proportionality between an increase in cell number in the epidermis and an increase in sepal length. The role of the two hormones in increasing epidermal cell length in sepals was distinct and separate. Exogenous IAA had no effect upon cell division but was required for cell elongation, while GA was required for cell division and had no effect on cell elongation. The GA effect in promoting cell division in the sepals was substantiated by use of autoradiography. If the buds were grown on media with GA, twice as many epidermal cells along the central file incorporated significant amounts of tritiated thymidine. The cell cycle of the epidermal cells of the GA-treated sepals was ca. 8.7 hr in duration and ca. 13.0 hr if GA was deleted from the medium.     

Whitebellied sunbirds (<Emphasis Type="Italic">Nectarinia talatala</Emphasis>, Nectariniidae) do not prefer artificial nectar containing amino acids

Amino acids are the most abundant class of compounds in nectar after sugars. Like its sugar concentration, the amino acid concentration of nectar has been linked to pollinator type, and it has been suggested that amino acid concentrations are high in the floral nectars of plant species pollinated by passerine birds compared to those pollinated by hummingbirds. We investigated the feeding response of whitebellied sunbirds (Nectarinia talatala) to the inclusion of amino acids in artificial nectar (0.63 M sucrose solution). The response to asparagine, glutamine, phenylalanine, proline, serine and valine, amino acids commonly found in floral nectars, was tested individually and using a mixture of all six amino acids, at two different concentrations (2 and 15 mM). Sunbirds showed no significant preference for amino acids in nectar, or avoided them, especially at the higher concentration. We discuss these findings in the light of the nitrogen requirements of nectarivorous birds and data on amino acids in floral nectars.     

Changes in proline level in response to osmotic stress and exogenous amino acids in Raphanus sativus L. seedlings

The changes in endogenous proline levels of Raphanus sativus L. seedlings was monitored in presence of exogenous amino acids in normal and osmotically stressed seedlings. In unstressed seedlings, proline uptake was detected only at higher (1 mM) concentration of applied L-proline. however, proline uptake was promoted at all (1 μM to 1000 μM) concentrations of applied L-proline under osmotic stress conditions. Amongst other exogenous amino acids, L-leucine, L-glutamic acid, L-alanine, and L-histidine enhanced endogenous levels of proline, while exogenous hydorxyproline and γ-amino butyric acid reduced it.     

THE FREE AMINO ACIDS OF HUMAN SPINAL FLUID DETERMINED BY ION EXCHANGE CHROMATOGRAPHY

(1) The free amino acids in human CSF from eighteen subjects have been determined. The analyses were performed on 0-75 ml of CSF by an ion exchange chromatographic method which is capable of detection to the 10^?10 mole level. (2) The amino acids always found in readily detectable amounts were: taurine, threonine, serine, glutamine, glutamic acid, citrulline, glycine, alanine, α-NH₂-n- butyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ethanolamine, ornithine, lysine, histidine and arginine. Urea was present. Aspartic acid and cystine, though always present, occurred in small or trace amounts. Proline was found in four cases and tryptophan in thirteen cases. In addition, twelve unknown peaks were nearly always evident in every chromatogram. (3) Filtrates 10 times more concentrated than those used regularly were prepared from pooled CSF and analysed. These analyses clearly confirmed the presence of those amino acids which were normally in very low concentration and they also served to distinguish the twelve unknown compounds from confusion with baseline artifacts. (4) The distribution of free amino acids in CSF was different from their distribution in blood plasma. (5) Despite a variety of neurological conditions and a wide age span few marked deviations were found in any of the amino acid concentrations.     

Free Amino Acids in the Leaves of Salvinia natans and Azolla filiculoides Grown in Light and Dark

Free amino acids were determined quantitatively by thin-layer chromatography and identified by various chromatographic methods in the leaves of two floating water ferns. Special interest was paid to the amount of γ-amino butyric acid. Water soluble nitrogen compounds increase in the leaves of both plants during the growth of three to four weeks in darkness. Compounds especially accumulating are ammonia and the amides asparagine and glutamine. The amount of most proper amino acids is reduced in the dark, except γ-amino butyric acid, which, in addition to amides, is the main amino compound increasing in darkness. 0.1 M HCl extracts the greatest amount of nitrogen compounds of low molecular weight from the dried leaves of the experimental plants. If we denote the amount of the dissolved amino compounds as 100 per cent, we can say that water extracts 96 per cent of the amino compounds, and that an 80 per cent ethanol solution extracts 79 per cent of these compounds.     

MOBILIZATION OF SYNAPTIC MEMBRANE-BOUND CALCIUM BY ACIDIC AMINO ACIDS1

A fluorescent chelate probe (chlorotetracycline) and radioactive ⁴⁵Ca were used to study the effects of amino acids on the calcium bound to external synaptosomal membranes isolated from guineapig brain. Acidic amino acids released some of the membrane-bound calcium. On the basis of ⁴⁵Ca studies, the order of mobilization potency-DL-homocysteic acid and l -cysteic acid > l -aspartic acid, l -glutamic acid, d -glutamic acid > N-methyl-dl -glutamic acid and dl -cyteic acid-is in general agreement with that found by fluorescent chelate method with the exception of N-methyl-dl -aspartic acid and N-methyl-dl -glutamic acid, which are at least as potent as dl -homocysteic acid. This order of potency is observed only with a fraction enriched in external synaptosomal membranes, but not with microsomes, myelin and mitochondria. Neutral and basic amino acids, including glutamine. glycine and γ-aminobutyric acid are ineffective. These results suggest that acidic amino acids have a specific ability to mobilize membranebound calcium; this is consistent with the proposed role of some of these compounds as excitatory transmitters in the central nervous system.     

FREE AMINO ACID CHANGES DURING GERMINATION OF TELIOSPORES OF THE WHEAT BUNT FUNGUS,TILLETIA CARIES

Teliospores were aerated and agitated in a mineral salts medium and their free amino acid contents were analyzed at eight different times, from shortly after imbibition of water until just before germ tube emergence. In addition to the common amino acids, eight unidentified ninhydrin-positive components were detected. About 50 % or more of nearly each of the amino acids diffused out of the spores during the initial phase of germination. These released amino acids were actively taken up by the spores during the latter stages of germination. The free amino acids in largest amounts in the dormant spores of T. caries were arginine 15.0, glutamic acid 6.3, and alanine 3.7 μmoles per g dry spores. Together these three amino acids accounted for about 71 % of the total free amino acids in dormant spores of T. caries and T. controversa. The total amounts of free amino acids in spores of common bunt were much higher than in spores of dwarf bunt.     

INFLUENCE OF CERTAIN PLANT GROWTH REGULATORS AND CROWN-GALL RELATED SUBSTANCES ON BUD FORMATION AND GAMETOPHORE DEVELOPMENT OF THE MOSS PYLAISIELLA SELWYNII

Bud formation and gametophore development were studied in the moss Pylaisiella selwynii (Kindb.) Steere and Anderson grown from spores in a liquid medium consisting of inorganic salts. Indoleacetic acid and ethrel increased bud formation within a narrow concentration range. Copious bud formation was obtained with the five cytokinins tested at concentrations varying from 5 X 10⁻⁶ to 5 X 10⁻¹⁴ M. Except for about 10 % of the buds obtained with 6-γ, γ-dimethylallylaminopurine at 5 times 10⁻¹⁴ m, the cytokinin-induced buds failed to develop into normal gametophores. Octopine, lysopine, and octopinic acid, substances obtained from crown-gall tumors, increased bud formation at 10⁻³ m. On lysopine-treated plants these buds developed into typical gametophores. Gemma-like structures were obtained with octopine but no gametophores. l -arginine and l -lysine, the amino acids which respectively occur in octopine and lysopine, failed to induce gametophore formation although buds were obtained with 10⁻³ m lysine. γ-Guanidinobutyric acid induced bud formation at 10⁻³ m, but these buds developed into highly abnormal gametophores. The failure of buds obtained with many of these treatments to develop into gametophores appeared to result from the formation of new cell walls in other than the normal geometrical relationship during initial divisions of the pro-bud. The relevance of the findings to the crown-gall problem is discussed.     

TRANSPORT OF AMINO ACIDS BY THE SOIL ALGA STICHOCOCCUS BACILLARIS1

The green alga Stichococcus bacillaris Naeg. is able to take up at least eleven amino acids. All of these except glutamic and aspartic acids are transported by carrier systems that obey saturation kinetics. The acidic amino acids enter the cell by passive diffusion. Michaelis-Menten parameters (K_s and V_max) were calculated for several amino acids. All obey simple Michaelis-Menten behavior except for 2-methylalanine and leucine which may have double carrier systems of different affinities. Interactions between pairs of amino acids suggest that there is at least one carrier system specific for basic amino acids and probably several systems specific for neutral amino acids. Further analysis of neutral amino acid interactions reveal that the uptake of several amino acids is incompletely inhibited by competitor uptake at infinite concentration. The simplest interpretation of the data is the operation of three carrier systems for neutral amino acids, one of which has higher affinity and broader specificity than the other two. The amino acid carrier systems appear to operate by an active mechanism. The metabolic poison DCCD inhibits uptake up to 99%. The capacities of the neutral amino acid carrier systems are increased when cells are grown in medium containing suboptimal concentrations of nitrogen.     

Reduced nitrogen influences somatic embryo quality and plant regeneration from suspension cultures of orchardgrass

Summary Production of somatic embryos in suspension cultures ofDactylis glomerata L. (orchardgrass) was stimulated by the addition of various compounds containing reduced nitrogen to Schenk and Hildebrandt (SH) basal medium. Equimolar concentrations of combinations of proline and either serine or threonine supported embryogenesis, whereas, these amino acids individually did not promote embryogenesis. SH medium supplemented with 6 to 25 mM ammonium ion or Murashige-Skoog basal medium also supported embryo production. Ammonium ion did not act synergistically with either proline or serine to enhance embryogenesis. Embryos produced in media containing amino acid combinations were formed singly, did not exhibit secondary embryogenesis, and had significantly higher conversion rates compared to those formed in either SH medium supplemented with ammonium ion only or in combination with amino acids.     

THE EFFECTS OF GIBBERELLIC ACID AND A GROWTH RETARDANT ON OVULE FORMATION AND GROWTH OF EXCISED PISTILS OF NIGELLA RANUNCULACEAE

The effects of gibberellic acid (GA₃) and the growth retardant AMO-1618 on ovule formation in excised pistils of Nigella sativa L. were studied by sterile culture techniques. Gibberellic acid promoted pistil growth and inhibited ovule formation. The role of endogenous gibberellins in ovule formation and pistil growth was investigated by adding AMO to the basal medium. Both pistil lengths and ovule formation were reduced significantly with increasing concentrations of AMO. The addition of low concentrations of GA₃ to the medium restored pistil growth but did not reverse the inhibitory effect of AMO on ovule formation. The addition of kinetin or indoleacetic acid (IAA) to the medium containing AMO had no effect on pistil lengths. However, with the addition of 10⁻⁷ m kinetin, the number of ovules in pistils was increased but not to the levels found in pistils grown in the absence of AMO.     

EFFECTS OF EXOGENOUS AMINO ACIDS ON THE DEVELOPMENT OF MARCHANTIA POLYMORPHA GEMMALINGS

The effects of various nitrogenous compounds in concentrations ranging from 10^-4 to 10^-3 m on the growth and morphology of gemmae of Marchantia polymorpha L. were investigated under axenic conditions. Only nine of the 24 compounds tested were effective in altering the normal developmental pattern of this plant. l -Isoleucine, l -leucine, l -methionine or l -threonine resulted in a disruption of the apical regions at concentrations of 10^-3 m . The addition of lower concentrations of l -arginine, l -histidine, l -hydroxyproline, l -lysine or l -tryptophan to the nutrient medium resulted in morphological irregularities. These latter abnormal growth patterns were highly specific for the individual amino acids and ranged from lack of air chamber development to complete thallus disorganization. Both the formation and duration of abnormal developmental patterns were dependent upon the concentration of the amino acids employed. Although the experimental results are inconsistent with certain physiological mechanisms such as a general nitrogen toxicity, alternative possibilities are discussed.     

THE ANTIBACTERIAL SUBSTANCES OF THE MARINE ALGA STICHOCHRYSIS IMMOBILIS (CHRYSOPHYTA)12

Two substances with antibacterial activity have been concentrated from the culture medium of Stichochrysis immobilis before senescence occurred, and purified, by a Sephadex G-25 filtration followed by 2-dimensional thin-layer chromatography on Merck F-254 silica gel plates. One of the substances is obviously a peptide; 11 different amino acids are linked in a 30 amino acid molecule (MW 3400). It was not possible to assign a chemical group to the second substance (MW ? 2600); it may be related to “humic acids” (Kalle's gelbstoff). Growth of the most sensitive bacteria was completely inhibited by 20-fold, or higher, concentrations of substances from the algal culture filtrate (assuming no loss of substances during the concentration processes). The 54 strains of test bacteria were of various origins, though mostly isolated from algal cultures or obtained directly from the marine environment.     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

生长素极性运输抑制剂(TIBA)对烟草离体花柄花芽分化的影响

在只含6-BA(2mg/L)的MS培养基上,烟草花柄外植体形态学基端膨大,上着生再生花芽,而花柄中部大多都形成愈伤组织。添加IAA(2,10,20 mg/L)后,花柄基端膨大的现象依然存在,但再生花芽的分布并不限于基端,在花柄中部、顶端都可见再生花芽。花柄外植体中部愈伤组织的形成也随添加的IAA和IAA浓度升高而受到抑制。在上述培养基中添加生长素极性运输抑制剂TIBA后,无一花柄中部能形成愈伤组织,再生花芽的形态变化也很大,有具锥形花柄的花芽、喇叭叶和一些难于确定由何种器官衍生而来的喇叭状器官。这些异于正常形态的器官发生,显然与花柄外植体中生长素极性运输受抑制有关,本文对它们的形成机理作了一些推测。     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro II. Effects of Growth Regulators

Correlative effects between growth regulators added to a mediumand different physiological states of explants on adventitiousbud formation and flowering were investigated using Toreniastem segments cultured in vitro. Indoleacetic acid stimulatedfloral bud formation and its development in explants taken fromreproductive plants. These stimulative effects were clearlyseen in explants taken from plants in which flower abscissionwas taking place, but insignificant when explants were preparedfrom younger materials. Abscisic acid acted in a reverse wayto auxin, greatly promoting floral bud initiation and floweringof originally vegetative explnts. Zeatin at a concentrationof 1 mg/liter inhibited floral bud formation, and at a low concentrationsit was generally ineffective. However, floral bud formationand flowering of explants taken either from basal parts of stemsor from 18- to 20-week-old plants were promoted by zeatin treatment.The action of gibberellic acid seemed rather indirect: at aconcentration of 0.01 mg/liter, it generally stimulated floralbud formation but at a concentration of 1 mg/liter, it was ofteninhibitory. (Received January 17, 1981; Accepted February 21, 1981)