Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

Accumulation and detoxification of cadmium in <Emphasis Type="Italic">Brassica pekinensis</Emphasis> and <Emphasis Type="Italic">B. chinensis</Emphasis>

The effect of excessive Cd on the growth and metal uptake by leafy vegetables Brassica chinensis L. (cv. Wuyueman) and Brassica pekinensis (Lour.) Rupr. (cv. Qingyan 87-114) were studied in hydroponic solution culture. The Cd concentration higher than 10 μM significantly decreased the root elongation and leaf chlorophyll contents of both plant species. The shoots of B. pekinensis had significantly higher concentrations of total and water-soluble Cd than B. chinensis. The roots of both species accumulated more Cd than the shoots in all the Cd treatments. Most of the Cd in the roots was found in the cell walls. The shoot/root ratio of Cd concentrations in B. pekinensis was always greater than that in B. chinensis. But, the concentration and proportion of Cd in the cell walls in B. chinensis were higher than that in B. pekinensis. Cadmium treatments also increased the concentrations of total non-protein thiols (NPT) in the shoots of the both species. A significant correlation was found between the concentrations of soluble Cd and NPT in plant shoots.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Cumulative effect of low and high atrazine concentrations on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Atrazine belongs to the widely used herbicides blocking the electron transport chain in chloroplasts, thus resulting in the generation of active oxygen species. In the present work, we demonstrated that, at low concentrations mimicking residual amounts, atrazine enhanced the susceptibility of Arabidopsis plants to further treatments with the same herbicide applied at the recommended field rate. Arabidopsis thaliana plants were treated three times (at five-day intervals) with 1 µM atrazine. Five days after the last treatment, the plants were sprayed with 5 mM atrazine. Atrazine increased the levels of lipid peroxidation products, hydrogen peroxide, and ion leakage, and caused changes in the activities of antioxidant enzymes, such as superoxide dismutase, guaiacol peroxidase, and catalase.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 243–249.Original English Text Copyright © 2005 by Ivanov, Alexieva, Karanov.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Plant regeneration from <Emphasis Type="Italic">Gossypium davidsonii</Emphasis> protoplasts <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8^th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 10⁵ cm⁻³ was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40^th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 μM gibberellic acid.     

Indonesian Kickxellales: two species of <Emphasis Type="Italic">Coemansia</Emphasis> and <Emphasis Type="Italic">Linderina</Emphasis>

Four species belonging to Kickxellales (Kickxellomycotina) isolated from soil of Indonesia are described and illustrated. Two new species of Coemansia, C. asiatica and C. javaensis, were discovered in South Sulawesi and West Java, and two known species of Linderina, L. pennispora and L. macrospora, were discovered in East Kalimantan and South Sulawesi, respectively. These four species are newly added to the Indonesian mycobiota. A technique for inducing sporulation of C. javaensis and L. macrospora by adding substances derived from invertebrates such as aphids, nereids, or cladocerans to culture media is described.     

Endocellular aminopeptidase from <Emphasis Type="Italic">Astasia longa</Emphasis>

A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45°C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase.     

Characterization and <Emphasis Type="Italic">in vitro</Emphasis> expression patterns of extracellular degradative enzymes from non-pathogenic binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-G

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source -1,3 and -1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The -(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.     

Diverse dextranase genes from <Emphasis Type="Italic">Paenibacillus</Emphasis> species

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M_r of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M_r of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.     

<Emphasis Type="Italic">Pluteus magnus</Emphasis> and <Emphasis Type="Italic">Pluteus podospileus</Emphasis> f. <Emphasis Type="Italic">podospileus</Emphasis>, two agaric species new to Japan

Two taxa of the genus Pluteus, i.e., Pluteus magnus and Pluteus podospileus f. podospileus, are newly recorded from Japan. The macroscopic and microscopic features of these two species are described and illustrated.     

Biodecolourization of azo and triphenylmethane dyes by <Emphasis Type="Italic">Dichomitus squalens</Emphasis> and <Emphasis Type="Italic">Phlebia</Emphasis> spp

Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium. Journal of Industrial Microbiology & Biotechnology (2002) 28, 201–203 DOI: 10.1038/sj/jim/7000222 Received 12 July 2001/ Accepted in revised form 22 October 2001     

Post-pollination changes in the floral organs of two <Emphasis Type="Italic">Cymbidium</Emphasis> species

It was observed that the unpollinated flowers of Cymbidium pendulum (Roxb.) Sw. and C. aloifolium (L.) Sw. stayed fresh for 20 and 18 d, respectively, but attained senescence in 8 and 7 d, respectively, after pollination. The higher content of total soluble sugars, reducing sugars and free amino acids was observed in all the floral organs of pollinated flowers than in unpollinated ones. Pollination also up-regulated the activity of hydrolytic (α-amylase, β-amylase, invertase) and proteolytic enzymes (proteases) in floral organs. Amongst floral organs, the lip and perianth possessed highest contents of metabolites. Application of auxin inhibitor (0.25 μM triiodobenzoic acid) and ethylene inhibitor (0.25 μM AgNO₃) to the pollinated flowers partially prevented the process of senescence.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Response of antioxidant activity to excess copper in two cultivars of <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino

Changes in ascorbic acid content and antioxidant enzyme activities were investigated in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves of ‘Wutacai’ and ‘Erqing’ exposed to excess copper (Cu). Cu treatment reduced the fresh weight of shoot and root by 57% and 46% in ‘Wutacai’, and 60 and 54% in ‘Erqing’, respectively. The accumulation of copper in leaves was higher in ‘Wutacai’ than that in ‘Erqing’. Compared to the control, ascorbic acid (AsA) contents were significantly decreased after copper treatment in both cultivars, while they were higher in ‘Wutacai’ than in ‘Erqing’, which may explain the higher copper-tolerance of ‘Wutacai’ with higher copper accumulation. The higher AsA contents of ‘Wutacai’ resulted from their lower activities of degrading enzymes, such as ascorbate oxydase (AAO) and ascorbate peroxidase (APX), as well as the increasing activity of dehydroascorbate reductase (DHAR) after copper treatment compared with ‘Erqing’. Copper stimulated superoxide dismutase (SOD) activity in both cultivars, but for catalase (CAT), there was little difference between both cultivars. Peroxidases (POD) activity was decreased after copper treatment in ‘Erqing’, while in ‘Wutacai’, it was significantly increased at 14 days, and POD activity was higher in ‘Wutacai’ than that in ‘Erqing’ at 21 and 28 days. Therefore, the induced increasing activity of POD in ‘Wutacai’ also played an important role in its copper tolerance.     

The cellulose synthase (<Emphasis Type="Italic">CESA</Emphasis>) gene superfamily of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

The CESA gene superfamily of Arabidopsis and other seed plants comprises the CESA family, which encodes the catalytic subunits of cellulose synthase, and eight families of CESA-like (CSL) genes whose functions are largely unknown. The CSL genes have been proposed to encode processive β-glycosyl transferases that synthesize noncellulosic cell wall polysaccharides. BLAST searches of EST and shotgun genomic sequences from the moss Physcomitrella patens (Hedw.) B.S.G. were used to identify genes with high similarity to vascular plant CESAs, CSLAs, CSLCs, and CSLDs. However, searches using Arabidopsis CSLBs, CSLEs, and CSLGs or rice CSLFs or CSLHs as queries identified no additional CESA superfamily members in P. patens, indicating that this moss lacks representatives of these families. Intron insertion sites are highly conserved between Arabidopsis and P. patens in all four shared gene families. However, phylogenetic analysis strongly supports independent diversification of the shared families in mosses and vascular plants. The lack of orthologs of vascular plant CESAs in the P. patens genome indicates that the divergence of mosses and vascular plants predated divergence and specialization of CESAs for primary and secondary cell wall syntheses and for distinct roles within the rosette terminal complexes. In contrast to Arabidopsis, the CSLD family is highly represented among P. patens ESTs. This is consistent with the proposed function of CSLDs in tip growth and the central role of tip growth in the development of the moss protonema. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Accession numbers: DQ417756, DQ417757, DQ898284–6, DQ898147–54, DQ902545–51.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.