Microspore development in cultured maize anthers

The present study follows in vivo and in vitro microspore development utilizing an anther culture-responsive maize genotype (Pa91×FR16) and a DNA-specific fluorescent dye (mithramycin). Cultured anthers were sampled at various times and scored for abnormal microspore divisions, multicellular masses, and embryo-like structures. The frequency of abnormal microspore divisions reached a peak during the first 7 days in culture and then declined. The vegetative nucleus was mitotically active in culture with over 50% of the induced microspores exhibiting this type of division. Multicellular masses and embryo-like structures first appeared in the 14 and 25 day samples, respectively. Most of the microspores did not reach the multicellular stage and an even greater mortality occurred during the formation of embryo-like structures.     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

Promiscuous,non-catalytic,tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>) plants

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.     

Storage in water ofRhizobium phaseoli cells harvested from continuous culture and its effect on biological nitrogen fixation

Summary Taken cells from cultures ofRhizobium phaseoli grown at different dilution rates were tested for plant growth promotion in a bioassay withPhaseolus vulgaris, before and after storage in water for one month. Cells grown at higher dilution rates showed a greater loss of effectiveness     

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots,plantlets and seedlings of Pinus pinaster

The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinaster were compared respectively with those of shoots collected from seedlings and whole plants.The usefulness of the nitrate of the medium used for in vitro axillary shoot formation is demonstrated by the occurrence of initial NR activity in the explants. When fed in a non in vitro situation with a 50 mM KNO₃ solution, they have the same induced capacity to reduce nitrate as do shoots from seedlings, even though the latter accumulate less nitrate. Plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings. In both types of plants, the Nitrate Reductase potential is greater in roots than in shoots.Abbreviation NR Nitrate Reductase - BA 6-Benzyladenine     

Transformation of Bacillus subtilis by electroporation

Summary Optimal conditions for the transformation of Bacillus subtilis by electroporation were achieved. Frequencies of greater than 10⁵ transformants/&#x03BC;g of plasmid DNA were obtained for a number of strains and plasmids. Increased transformation efficiency of mini-prep DNA from B. subtilis and Escherichia coli was obtained after microdialysis.     

The absence of direct effects of prostaglandin F2α on preimplantation mouse embryos in vitro

Preimplantation mouse embryos were cultured in vitro in concentrations of PGF_2α ranging from 5 nanograms/ml to 100 micrograms/ml. Embryos were cultured at the 2-cell stage, 4-cell stage, 8-cell stage and the morula stage and observed for normal development to the blastocyst stage. There was no significant difference (P < .1) between the number of blastocysts attained in the control group and those in the experimental groups. After reaching the blastocyst stage, the embryos were transferred to the uteri of pseudopregnant recipient females. Again, there was no significant difference (P < .3) between controls and PGF_2α treated embryos in the number developing to term. These data suggest that exposure to PGF_2α does not result in teratogenic effects or early death in preimplantation embryos.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Extraction of bovine serum albumin with reverse micelles from glucosylammonium and lactosylammonium surfactants

Reverse micelle extraction is still in the stage of laboratory. Major limitation associated with use of synthetic surfactants in reverse micelle extraction process is the unfolding or denaturation of proteins. Sugar surfactants are thought non-toxic and environmentally benign, and can exhibit interesting interfacial properties, but the application of sugar-based surfactants in protein extraction is still limited. In the present study, we extracted bovine serum albumin (BSA) by using reverse micelles from glucosylammonium (GA) and lactosylammonium (LA) surfactants (with dicarboxylate as counter ion). It was found that under optimum condition, (1) the maximum forward extraction efficiency was ca. 86% with GA, while only around 50% with LA, and (2) almost all BSA solubilized in reverse micelles prepared from GA could be recovered into aqueous phase, while the recovery of BSA from the reverse micelles of LA was lower. In addition, the optimum extraction parameters were closely related to surfactant structure. Therefore, the electrostatic interaction, H-bonding and sugar head size should be important for BSA transfer.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Summary Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l^–1 KH₂PO₄ and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.     

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

Increasing the variability of Lycopersicon peruvianum Mill. by protoplast fusion with Petunia hybrida L.

Somatic hybridization of Lycopersicon peruvianum and Petunia hybrida was carried out to transfer cytoplasmic male sterility from Petunia to Lycopersicon. Cytological, morphological and biochemical analyses were performed to characterize the regenerated plants. Two regenerated plants, R₃ and R₆, were male sterile. R₃ possessed chromosomes morphologically similar to those of both parental types. Leaf morphologies of these two plants and a third plant, R₇, were intermediate between the two parents. The stability of RUBPCase was verified during parental plant development and after in vitro culture. Plant R₇ presented a new form of the large subunit of RUBPCase.     

Extracellular L-glutaminase production by marine bacteria

Summary Four species of bacteria which includedPseudomonas fluorescens,Vibrio cholerae andVibrio costicola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6–6.8 times greater than the intracellular production by all the tested strains.     

Cadmium bio-sorption by bacterial exopolysaccharide

Summary Bacterial exopolysaccharides can significantly enhance bio-sorption of cadmium. Exopolysaccharide fromArthrobacter viscosus has a 2.3 times greater accumulation capacity than the equivalent weight of intact cells and is 13.7 times more effective than the cells ofArthrobacter globiformis, an organism that does not produce exopolysaccharide.     

Kinetics of alcohol production by zymomonas mobilis at high sugar concentrations

Summary Studies on the growth ofZ.mobilis revealed that high concentrations of glucose (10-25%) can be efficiently and rapidly converted to ethanol in batch culture. By comparison withS. carlsbergensis,Z.mobilis had specific glucose uptake rates and specific ethanol productivies several times greater than the yeast.Z.mobilis also had ethanol yields of up to 97% of a theoretical value.     

SPORTS1.0: A Tool for Annotating and Profiling Non-coding RNAs Optimized for rRNA- and tRNA-derived Small RNAs

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users’ input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.