Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosome-inactivating protein b-32

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn     

Optimal strategy of plant antiherbivore defense: Implications for apparency and resource-availability theories

Plants produce chemicals as methods against animal herbivory. Such chemical defenses are classified into two major categories: (i) quantitative defenses with massive production of indigestible substances; and (ii) qualitative defenses with production of poisonous substances. A mathematical model was developed that identified factors that favored the evolution of quantitative defenses. Selecting an annual plant for simplicity, the allocation of photosynthetic production between growth substances and defense substances was considered. If the plant invests more in defense substances, it can protect itself more efficiently from herbivory but with a reduced growth rate. If it invests more in growth substances, the contrary holds. Using Pontoryagin's maximum principle, the following results were obtained: (i) the plant should conduct quantitative defenses when the growth rate (G), reflecting resource-availability, is low and the growth period (T) is long as well; (ii) if the plant invests in quantitative defenses, the optimal proportion of defense substances (χ^*) should be higher asG is smaller, but it is independent ofT; and (iii) the value of χ^* is not monotone for the effectiveness of defense substance (A), but has a maximum at an intermediate value ofA. Predictions of the model partly supported both Feeny's apparency theory, claiming that apparent plants or their parts for herbivores should quantitatively defend themselves, and Coley's resource-availability theory, claiming that plants with rich resources should invest in growth rather than defense.     

Recent advances in plant cell cultures in bioreactors

Two key issues in the application of plant-cell-culture technology to the production of valuable secondary metabolites are reviewed: the selection of cell lines with suitable genetic, biochemical and physiological characteristics; and the optimization of bioreactor environments. Although great progress has been made in recent years in the design, selection and optimization of bioreactor hardware, optimization of environmental factors such as medium components, light irradiation and O₂ supply needs detailed investigations for each case. With a better understanding of plant cell metabolism and physiology, further developments in cultivation processes, such as process integration and on-line monitoring and control, can be expected in the near future.J.-J. Zhong and J.-T. Yu are with the Research Institute of Biochemical Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China T. Yoshida is with the International Center of Cooperative Research in Biotechnology (ICBiotech), Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Die Anwendung einer ‘deduktiven Methode syntaxonomischer Klassifikation’ bei der Bearbeitung der strassenbegleitenden Pflanzengesellschaften Nordostböhmens

Zusammenfassung In der vorgelegten Arbeit wurde die sogenante deduktive Klassifikationsmethode der anthropogenen Pflanzengesellschaften behandelt. Diese Methode geht vom System der höheren Vegetationseinheiten der Braun-Blanquet Schule aus. Sie ermöglicht die syntaxonomische Bearbeitung zahlreicher anthropogener Gesellschaften unserer Kulturlandschaft, die nur aus Arten mit breiterer ökologischer und zönologischer Amplitude zusammengesetzt sind, und zwar ohne unerwünschte Verwechslung der Bestimmungsmerkmale einzelner Rangstufen des Systems (Klassen-, Ordnungs-, Verbands- und Assoziationskenn-und trennarten). Die Grundprinzipien der Methode wurden schon früher von Kopecký & Hejný (1971, 1973, 1974) und Kopecký (1976) erläutert. Die vorliegende Arbeit bringt eine präzisierte Auslegung der Methode auf Grund neuer Erfahrungen, die bei der Bearbeitung der spontan entstandenen strassenbegleitenden Gesellschaften derMolinio-Arrhenatheretea Tx. 1937 in Nordostböhmen gewonnen wurden. Aus dem reichen Aufnahmematerial werden nur einige Aufnahmen der Basalgesellschaften mitAlopecurus pratensis und der von ihnen abgeleiteten Derivatgesellschaften mitDactylis glomerata undAgropyron repens fur die anschauliche Demonstration der Methode beigegeben (Tab. 1 und 2). Die Aufnahmen der übrigen Gesellschafts-typen werden in einer anderen Arbeit (Kopecký 1978) veröffentlicht.

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Summary Use of a so-called deductive method in syntaxonomic classification of roadside plant communities in NE Bohemia.The present paper explains the so-called deductive method of syntaxonomic classification of plant communities, derived from the system of syntaxa of higher rank of the Braun-Blanquet approach. This method enables the synthesis of vegetations units of the varied anthropogenic plant communities in the man-made landscape, and their arrangement within the existing system, without changing the classification and determination criteria of both the higher and lower syntaxa in this system. Basic principles of this method have been outlined earlier by Kopecký & Hejný (1971, 1973, 1974) and Kopecký (1976). Following the newly obtained data and experience, this paper brings further details and more precise explanation of the method. Recently, the deductive method has been applied to the classification of turf communities which spontaneously develop along the roads in NE Bohemia. In order to illustrate the principles of the method, selected relevés of basal communities withAlopecurus pratensis (Table 1), and respective communities derived from these basal communities (Table 2) are presented. A complete set of vegetation relevés of all other roadside communities will be published elsewhere (Kopecký 1978).

Die wissenschaftlichen Pflanzennamen sind nach Rothmaler et al. (1966) angeführt.     

Action spectra for the light inhibition of flowering and its reversal inLemna paucicostata T-101

Lemna paucicostata Hegelm. T-101, a short-day plant, flowers when plants preirradiated with red light (R) for 24 h are subjected to inductive darkness for 72 h followed by two short-day cycles (6 h R+ 18 h dark). However, flowering is inhibited by blue-or far-red-light pulses applied at the beginning of the inductive dark period. These inhibitory light effects are fully reversible by a R pulse. The action spectra for the inhibitory light effect and for its reversal show that the light pulses act exclusively through phytochrome. It is concluded that a low level of Pfr at the beginning of the inductive dark period prevents flowering.Abbreviations R red (light) - B blue (light) - FR far-red (light)     

Papyrus swamp development in the Upemba Basin, Zaïre: studies of population structure in Cyperus papyrus stands

The ecology and distribution of papyrus swamps are briefly reviewed together with the ecology, distribution structure and growth characteristics of Cyperus papyrus.
Three age-classes of culm are recognized and girth and density of each are recorded along transects through mree swamps in the Upemba Basin of Zaïre. A regression of culm-unit biomass on culm girth, developed for Ugandan swamps, is used to convert Upemba measurements into biomasses. It is shown that simple measurements, taken at only a single point in time, yield much information on both the structure and the dynamics of papyrus populations.
It is suggested that the standing-crops of such populations are determined by available light, and by local temperature regimes, and are therefore constant, irrespective of site quality (nutrient status), for any particular climatic zone. Culm-duration (life-time) is not significantly influenced by climate and the ratio culms/rhizomes provides a useful indicator of comparative productivity. Unlike community biomass, productivity can be limited by site quality.     

Estimation of growth yield and maintenance coefficient of plant cell suspensions

Methodology is presented for the determination of growth yield (Y(g)) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Y(g) and m requires measurements of specific growth rate (micro) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Y(g) and m because micro is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the K(m) for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the K(m), the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Y(g) and m because various steady-state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of micro and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters.