Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine     

Molecular analysis of the inheritance of the S-5 locus,conferring wide compatibility in Indica/Japonica hybrids of rice (O. sativa L.)

RFLP analysis was conducted on a population derived from a three-way cross to determine the location of the hybrid sterility locus, S-5, in relation to mapped molecular markers and to identify markers that would be useful for selection in breeding. S-5 is of interest to rice breeders because it is associated with spikelet sterility of F₁ hybrids in Indica/Japonica crosses. Identification of an S-5 allele which confers fertility in Indica/Japonica hybrids when introgressed into either the Indica or the Japonica parent has been reported. Varieties carrying this S-5 ⁿ allele are known as wide compatibility varieties (WCV). Our data suggests that RFLP marker RG213 on chromosome 6 is closely linked to the S-5 locus and can be efficiently used to identify wide compatibility (WC) lines. RG213 is a single-copy genomic clone that detects three bands of different molecular weights in DNA from Japonica (Akihikari) and Indica (IR36) varieties and WC line (Nekken 2). We demonstrate that the three alleles detected by this marker could be used to trace the inheritance of the wide compatible phenotype in breeders' material.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Somatic embryogenesis in callus culture of Mussaenda erythrophylla cvs. Queen Sirikit and Rosea

Somatic embryogenesis was achieved from mid-rib and internodal calluses of Mussaenda erythrophylla L. cvs. Queen Sirikit and Rosea cultured on Murashige and Skoog basal medium containing 8.9 M BA+0.57 M IAA+10 mg l^-1 ascorbic acid. Clumps of somatic embryos were separated and grown into complete plantlets when transferred to 1/2 MS medium+37 M adenine sulphate with 2% (w/v) sucrose.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani)

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F₄ bulked populations from a cross between the susceptible variety Lemont and the resistant variety Teqing, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the LemontxTeqing cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Breeding soybeans for the tropics capable of nodulating effectively with indigenousRhizobium spp.

Summary Most soybean varieties fail to nodulate effectively in tropical soils unless inoculated with a competitive strain ofRhizobium japonicum. Developing countries in the tropics, with few exceptions, lack inoculant industries to produce and distribute viable inoculants to small farmers and extension programs to teach them to use inoculant. Several soybean genotypes have been identified that nodulate effectively with many strains of the cowpea inoculation group which is ubiquitous in tropical soils of Africa. Soybean genotypes that nodulate and grow well without inoculant application are called promiscuous. Methodologies for incorporation of the promiscuity character into high-yielding backgrounds are discussed.Supported in part by grant 05-0560 from United Nations Development Program to the International Institute of Tropical Agriculture.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.