A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

THE ORIGIN AND EVOLUTION OF PARTHENOGENESIS IN HETERONOTIA BINOEI (GEKKONIDAE): EXTENSIVE GENOTYPIC DIVERSITY AMONG PARTHENOGENS

Variation at 18 allozyme loci was assayed among representatives of the geographically widespread, triploid parthenogenetic form of Heteronotia binoei. A minimum of 52 different genotypes were observed among 143 individuals. Virtually all localities sampled had multiple genotypes among the unisexuals. This represents unusually high genotypic diversity for a unisexual vertebrate. Heterozygosity in the triploids was higher than in diploid bisexual populations of H. binoei. Comparison with the alleles present in the diploid bisexuals confirms that the parthenogens are hybrids and indicates that most of the genotypic diversity stems from repetitive hybrid origins. However, the presence of some alleles unique to the parthenogens suggests that mutation adds to their genetic diversity. The genetic structure of this geographically widespread parthenogen suggests the hypothesis that the persistence and spread of the unisexual lineages is facilitated by genotypic diversity.     

Localisation of degradative enzymes in white-rot decay of lignocellulose

The use of immunogold-cytochemical labelling techniques in electron microscopy of wood infected by basidiomycete fungi has assisted in the elucidation of the localisation of enzymes which degrade lignocellulose. The use of specific immunocytochemical techniques is discussed with respect to the authenticity and accuracy of the methods, the use of adequate controls in the gold-labelling procedure, and the immunospecificity of the antibodies.Localisation of the lignin-degrading enzymes, lignin-peroxidase and laccase, has shown that these enzymes do not bind to wood cell walls unless the process of decay has already commenced. Similarly localisation of cellulases Endoglucanase II (EGII) and Cellobiohydrolase I (CBHI) has shown that these enzymes only bind to exposed ends of cellulose fibrils and to partially degraded areas of the wood cell wall. -Glucosidase is always immobilised within the extracellular polysaccharide layer surrounding fungal hyphae.This review postulates that there is regulation of the release sequence of these lignocellulolytic enzymes defining the spatial arrangement between the hyphae and the wood cell wall. This hypothesis is presented diagrammatically.     

<i>In vitro</i> Antibacterial Activity of <i>Moringa oleifera</i> Ethanolic Extract against Tomato Phytopathogenic Bacteria

The tomato (Solanum lycopersicum L.) is one of the world’s most important vegetable crops. Still, phytopathogenic bacteria affect the yield and quality of tomato cultivation, like Agrobacterium tumefeciens (At), Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst), Ralstonia solanacearum (Rs), and Xanthomonas axonopodis (Xa). Synthetic chemical products are used mostly on disease plant control, but overuse generates resistance to bacterial control. This study aimed to evaluate the in vitro antibacterial activity of the ethanolic extract of Moringa oleifera Lam. leaves against At, Cmm, Pst, Rs, and Xa, as well as information about this plant species’ chemical composition. Antibacterial activity against pathogens observed by microplate technique, phytochemical screening, and FTIR analysis revealed different bio-active compounds on ethanolic extracts with antibacterial activity. The growth inhibition rate ranged between 0.08% and 99.94%. The inhibitory concentration, IC50, required to inhibit 50% of At, Cmm, Pst, Rs, and Xa bacterial growth, was 276.67, 350.48, 277.85, 351.49, and 283.22 mg/L, respectively. Inhibition of phytopathogen bacteria’s growth increased as the concentrations of the extract also increased. Moringa oleifera extract can be recommended as a potent bio-bactericide.     

Fossil liberation: a model to explain high biodiversity in the Triassic Cassian Formation

With 1429 animal species, the Triassic Cassian Formation in the Dolomites, Southern Alps (Italy), yields the highest species richness reported from any spatially constrained pre-Quaternary formation known to science. The high preserved diversity is partly attributable to a high primary diversity governed by the tropical setting, increasing alpha diversity, and the breadth of habitats spurring beta diversity. More important is the excellent preservation of fossils and the ease with which they can be extracted from the poorly lithified sediments. We propose the term ‘liberation Lagerstätten’ to capture this preservational window. In contrast to conservation Lagerstätten, liberation Lagerstätten like the Cassian Formation originate from normal marine conditions but low-grade diagenesis. Molluscs contribute substantially to species richness, comprising 67% of all invertebrate species in the Cassian Formation. The gastropod dominance (39% of all species) is nearly as great as in Recent tropical settings, contradicting the concept of a substantial Cenozoic rise.     

Neuroactive diol and acyloin metabolites from cone snail-associated bacteria

The bacterium Gordonia sp. 647 W.R.1a.05 was cultivated from the venom duct of the cone snail, Conus circumcisus. The Gordonia sp. organic extract modulated the action potential of mouse dorsal root ganglion neurons. Assay-guided fractionation led to the identification of the new compound circumcin A (1) and 11 known analogs (2–12). Two of these compounds, kurasoin B (7) and soraphinol A (8), were active in a human norepinephrine transporter assay with K_i values of 2575 and 867 nM, respectively. No neuroactivity had previously been reported for compounds in this structural class. Gordonia species have been reproducibly isolated from four different cone snail species, indicating a consistent association between these organisms.     

The role of chick Ebf genes in the mediolateral patterning of the somites

This study was conducted to check whether the three chick Early B‐cell Factor (Ebf) genes, particularly cEbf1, would be targets for Shh and Bmp signals during somites mediolateral (ML) patterning. Tissue manipulations and gain and loss of function experiments for Shh and Bmp4 were performed and the results revealed that cEbf1 expression was initiated in the cranial presomitic mesoderm by low dose of Bmp4 from the lateral mesoderm and maintained in the ventromedial part of the epithelial somite and the medial sclerotome by Shh from the notochord; while cEbf2/3 expression was induced and maintained by Bmp4 and inhibited by high dose of Shh. To determine whether Ebf1 plays a role in somite patterning, transfection of a dominant‐negative construct was carried out; this showed suppression of cPax1 expression in the medial sclerotome and upregulation and medial expansion of cEbf3 and cPax3 expression in sclerotome and dermomyotome, respectively, suggesting that Ebf1 is important for ML patterning. Thus, it is possible that low doses of Bmp4 set up Ebf1 expression which, together with Shh from the notochord, leads to establishment of the medial sclerotome and suppression of lateral identities. These data also conclude that Bmp4 is required in both the medial and lateral domain of the somitic mesoderm to keep the ML identity of the sclerotome through maintenance of cEbf gene expression. These striking findings are novel and give a new insight on the role of Bmp4 on mediolateral patterning of somites.     

Cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation

Summary The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1β and tumor necrosis factor α induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor β had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.