The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

Plant regeneration from somatic embryos in black pepper

Embryogenic callus derived from zygotic embryos of black pepper (Piper nigrum Linn.) were induced to form somatic embryos on solid and liquid Schenk and Hildebrandt basal medium. Callus proliferation, somatic embryo-genesis and germination of embryos were achieved in about 8 months in static cultures while it took only 8 weeks in liquid suspension cultures. The highest number of embryos and plantlets was produced from cells grown as suspension cultures raised in half-strength medium without growth regulators and sucrose level reduced from 3% to 1.5%. Regenerated plants were established in soil.     

Bioreactor design for propagation of somatic embryos

Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (clonal or somatic seeds). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent quiet zones in the suspension in which cells can settle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24-h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (Daucus carota), Norway spruce (Picea abies), birch (Betula pendula), cyclamen (Cyclamen persicum) and shoot cultures of Christmas begonia (Begonia x cheimantha). The paper also discusses recommendations for improvements of the current system for future revisions.     

Control of growth and differentiation of bioreactor cultures of Physcomitrella by environmental parameters

The effect of physical and chemical environmental parameters on growth and differentiation of suspension cultures of the moss Physcomitrella patens in bioreactors was investigated. By supplementation of the aeration gas with 2% (v/v) CO₂ as well as by continuous illumination, growth of this photoautotrophic growing batch culture was markedly enhanced, resulting in a doubling time of 1.2d. The growth rate of semicontinuously growing bioreactor cultures was not affected by controlling the pH of the culture medium with set points at 4.5 or 7.0. However, growth of the culture at pH 7.0 resulted in increased caulonema development, thus showing a distinct effect on moss differentiation. The impact on research and plant biotechnological applications of the potential to control moss growth and differentiation by environmental parameters is discussed.     

Effects of oxygen partial pressure and combined nitrogen on N2-fixation (C2H2) associated withZea mays and other gramineous species

Summary The objectives of this investigation were to determine the effects of oxygen partial pressure (pO₂) and combined nitrogen (NH ₄ ⁺ ) on rates of acetylene reduction (AR) associated with roots of intact corn, sorghum, and pearl millet plants. Soil-grown plants were carefully removed from soil and incubated hydroponically with the root system enclosed in a plastic cylinder; the tops were left exposed to ambient conditions. Oxygen concentrations around the root systems were controlled by sparging the nutrient solution with known quantities of O₂ in N₂. Ammonium nitrogen was added to the nutrient solution following establishment of AR rates to determine its effect on rates of N₂-fixation (AR). Substantial AR rates (0.1–1.5 mol C₂H₄ g dry wt^–1 h^–1) were associated with roots exposed to 0–2% O₂ (v/v) (0.0–2.02 kPa) in N₂ following at 12–24 h period of exposure to the reduced oxygen tension. Root systems exposed to air failed to demonstrate AR while those exposed to 100% N₂ showed lower activity than those at reduced pO₂ values. Addition of NH ₄ ⁺ (10–20 g N ml^–1 of nutrient solution) reduced AR by 75–90% within 24 h after addition. Oxygen uptake by roots exposed to low pO₂ was substantially reduced.     

Comparative analysis of cell cycle events in zygotic and somatic embryos of Cyclamen persicum indicates strong resemblance of somatic embryos to recalcitrant seeds

Embryo development and germination of Cyclamen persicum have been comparatively characterized for zygotic and somatic embryos with regard to mitotic activity and morphology in order to identify developmental abnormalities in somatic embryogenesis. Zygotic embryo development proved to be highly synchronous with distinct periods of cell division, cell elongation and embryo maturation within a total period of 17 weeks of seed development. Somatic embryo development was accomplished within only 3 weeks, resulting in a mixture of morphologically highly variable embryos. No distinct developmental periods could be identified and no reduction of the mitotic activity was discovered for non-desiccated somatic embryos. Controlled desiccation of somatic embryos severely reduced their germination rate, demonstrating resemblance of somatic embryos to recalcitrant seeds, whereas zygotic Cyclamen seeds could be characterized as typically orthodox.     

Monitoring of haploid maize cell suspension culture conditions in bioreactors

Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.Abbreviations 2,4-d 2,4-dichlorophenoxyacetitc acid - NAA naphthalene acetic acid     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

Variable alteration of regional tissue oxygen pressure in rat hippocampus by acute swimming exercise

One of the events in the brain is an increasing cerebral blood flow during exercise. The tissue oxygen level may be increased because blood flow correlates with tissue oxygen level. However, it is little known whether the tissue oxygen pressure in hippocampal region (Hip-pO₂) will be affected by exercise.     

The effect of exercise intensity and duration on the oxygen deficit and excess post-exercise oxygen consumption

Nine males with mean maximal oxygen consumption (VO2max) = 63.0 ml.kg-1.min-1, SD 5.7 and mean body fat = 10.6%, SD 3.1 each completed nine counterbalanced treatments comprising 20, 50 and 80 min of treadmill exercise at 30, 50 and 70% VO2max. The O2 deficit, 8 h excess post-exercise oxygen consumption (EPOC) and EPOC:O2 deficit ratio were calculated for all subjects relative to mean values obtained from 2 control days each lasting 9.3 h. The O2 deficit, which was essentially independent of exercise duration, increased significantly (P less than 0.05) with intensity such that the overall mean values for the three 30%, 50% and 70% VO2max workloads were 0.83, 1.89 and 3.09 l, respectively. While there were no significant differences (P greater than 0.05) between the three EPOCs after walking at 30% VO2max for 20 (1.01 l), 50 (1.43 l) and 80 min (1.04 l), respectively, the EPOC thereafter increased (P less than 0.05) with both intensity and duration such that the increments were much greater for the three 70% VO2max workloads (EPOC: 20 min = 5.68 l; 50 min = 10.04 l; 80 min = 14.59 l) than for the three 50% VO2max workloads (EPOC: 20 min = 3.14 l; 50 min = 5.19 l; 80 min = 6.10 l). An analysis of variance indicated that exercise intensity was the major determinant of the EPOC since it explained five times more of the EPOC variance than either exercise duration or the intensity times duration interaction. The mean EPOC:O2 deficit ratio ranged from 0.8 to 4.5 and generally increased with both exercise intensity and duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

土壤氧气可获得性对双季稻田温室气体排放通量的影响

为探讨土壤氧气可获得性(SOA)对双季稻田温室气体排放的影响,利用静态箱气相色谱法对多种管理措施影响下稻田温室气体排放通量和土壤氧化还原电位(Eh)、pH值及田间淹水深度(H)等3种SOA因子进行了观测。结果表明,甲烷(CH₄)排放最集中的Eh值、pH值和H范围分别为-100-0mV、5 < pH < 6和1-5cm,3个范围内分别观测到48.8%、61.1%和77.0%的CH₄排放,其中H对CH₄排放影响最明显,单独由其就可解释37.8%的CH₄排放通量(P < 0.0001)。对于氧化亚氮(N₂O),观测到较多的负通量,其纯排放最密集的3种SOA因子的范围分别是:0-100mV、5 < pH < 6和1-5cm,而200-300mV是其排放的临界Eh范围,高于此范围N₂O排放极少。厌氧的反硝化过程是双季稻田N₂O产生的主导过程。可为水稻田温室气体排放机理研究提供基础数据。     

The types of bioreactors used for shoots and embryos

Plant regenerated organs such as shoots, bulbs, microtubers, corms, embryos, etc. have been successfully proliferated in the bioreactor. The use of a bioreactor leads to the development of technology suitable for large scale plant propagation. The basic construction and characteristics of various types of bioreactor systems are reviewed in relation to shoot and embryo cultures. A pilot scale 500 liter bioreactor system was applied to the production of large scale Stevia rebaudiana shoots.Abbreviations DW dry weight - EC electrical conductivity - FW fresh weight - ORP oxidation-reduction potential     

Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H₂O₂. The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2α kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34^cdc2 kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production

Design and experimental validation of advanced pO₂ controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO₂ in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO₂ control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.     

Control of cell proliferation during plant development

Knowledge of the control of cell division in eukaryotes has increased tremendously in recent years. The isolation and characterization of the major players from a number of systems and the study of their interactions have led to a comprehensive understanding of how the different components of the cell cycle apparatus are brought together and assembled in a fine-tuned machinery. Many parts of this machine are highly conserved in organisms as evolutionary distant as yeast and animals. Some key regulators of cell division have also been identified in higher plants and have been shown to be functional homologues of the yeast or animal proteins. Although still in its early days, investigations into the regulation of these molecules have provided some clues on how cell division is coupled to plant development.     

Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2

Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH(2) to thromboxane A(2) (TXA(2)), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA(2). The present results suggest that overexpression of TXS and subsequent excess production of TXA(2) in the cancer cells may be involved in the tumor growth of human colorectum.     

Significances of pH and temperature on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein, glycoprotein 96 (gp96), elicits both innate and adaptive immune responses against tumors or viral infections. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, significances of pH and temperature on the novel bioprocess were studied in stirred-tank bioreactor. Lowering of culture pH and temperature led to a significant reduction of average specific growth rate but cell viability remained high for a prolonged cultivation time resulting in a higher integral of viable cells. Both the maximal viable cell density and gp96 production were attained at a pH of 7.0. Interestingly, gp96 production was increased above and below 37 °C, presumably because gp96 biosynthesis was induced when MethA tumor cell underwent heat or cold. For MethA tumor cell growth 37 °C was desirable, while gp96 production and productivity was obtained at their peak values at 40 °C. The results of this work might be useful to scale-up the bioprocess into the pilot scale.