A study of mitochondrial-extramitochondrial interactions in giant tissue culture cells by microfluorimetry-microelectrophoresis

Histochemistry and Cell Biology -     

Oleate desaturation in young winter wheat root tissue

[1,2-¹⁴C]Acetate was incorporated into the lipids of young wheat (Triticum aestivum L. cv Kharkov 22 MC) root tissue, but predominantly into sterols. [1-¹⁴C]Ammonium oleate was initially incorporated mainly into phosphatidylcholine (PC), and later into triglycerides (TGs). Diglycerides (DGs) contained 16% of the lipid ¹⁴C after 5 minutes and 8% after 40 minutes. The proportion of the label of each lipid group incorporated into linoleate during an 80-minute incubation increased at similar rates for each group, and was always highest in PC. Radioactivity was detected in PC-linoleate earlier than in linoleate of the other groups. During a prolonged incubation after a 15-minute pulse labeling, the percentage of the lipid ¹⁴C incorporated into PC and DGs was high at the end of the pulse but decreased later, while that in TGs increased to 64% after 4 hours. The proportion of the label of each group recovered in linoleic acid peaked in all groups after 4 hours, except for the TGs where it increased slowly throughout the experiment. Only traces of radioactivity were detected in linolenate. The data are compatible with a pathway in which oleate is incorporated into PC, is desaturated to linoleate on PC, and where the linoleate-enriched DGs are transferred from PC to TGs.     

A soil-free root observation system for the study of root-microorganism interactions in maize

Background and aims

The root surface of a plant usually exceeds the leaf area and is constantly exposed to a variety of soil-borne microorganisms. Root pathogens and pests, as well as belowground interactions with beneficial microbes, can significantly influence a plants' performance. Unfortunately, the analysis of these interactions is often limited because of the arduous task of accessing roots growing in soil. Here, we present a soil-free root observation system (SF-ROBS) designed to grow maize (Zea mays) plants and to study root interactions with either beneficial or pathogenic microbes.

Methods

The SF-ROBS consists of pouches lined with wet filter paper supplying nutrient solution.

Results

The aspect of maize grown in the SF-ROBS was similar to soil-grown maize; the plant growth was similar for the shoot but different for the roots (biomass and length increased in the SF-ROBS). SF-ROBS-grown roots were successfully inoculated with the hemi-biotrophic maize fungal pathogen Colletotrichum graminicola and the beneficial rhizobacteria Pseudomonas putida KT2440. Thus, the SF-ROBS is a system suitable to study two major belowground phenomena, namely root fungal defense reactions and interactions of roots with beneficial soil-borne bacteria.

Conclusions

This system contributes to a better understanding of belowground plant microbe interactions in maize and most likely also in other crops.     

冬小麦根系分布规律

根据在郑州进行的冬小麦根系田间实测资料,研究了根长密度和根质量密度在砂壤土中的垂直分布.结果表明:冬小麦根量主要集中在上层,根长密度、根质量密度在0～50 cm土层内分别占57.7%和66.7%,而在50～100 cm层分别占23.4%和18.7%,根长密度和根质量密度随土壤深度的变化均符合指数函数形式;综合考虑根量分布、根系吸水等因素,确定了冬小麦适宜的底墒深度为100 cm.     

Tooth germ epithelial-mesenchymal interactions in tissue culture

The usefulness of the tooth germ in culture arises from the fact that it exemplifies those fundamental attributes of development, cell proliferation, cytodifferentiation, and morphogenesis, which we expect to find in the development of any metazoan organism. In culture, as in the organism, such development takes place in 3 dimensions. This study was undertaken to determine if it is possible to uncouple, by using 2 dimensions, cytodifferentiation from morphogenesis. Under the conditions used, cytodifferentiation in culture was not apparent (at the light microscope level). However, the following interesting observations were made: Cell populations arising from the same types of explants (enamel organ/enamel organ or dental papilla/dental papilla) readily flow together. Cell populations arising from dissimilar types of explants (enamel organ/dental papilla) form sharp boundaries at their interfaces. Additionally, cell populations arising from intact tooth germs differ from those arising from either enamel organs or dental papillae.     

Response of the root system of a winter wheat crop to waterlogging

This study was aimed at analysing and quantifying the response of the root system dynamics of a wheat crop to waterlogging. Two experiments were carried out in parallel: one under controlled conditions with semi-permanent water tables using lysimeters equipped with oxygen measurers, and the other under conditions of artificially drained plots by continually monitoring their hydraulic functioning. The root system was observed frequently using the root mapping method, and this made it possible to measure the growth of the root front, to estimate root densities, and infer growth indices from them. The results showed that the anoxic medium for wheat roots consisted of a water-saturated soil with an oxygen concentration of below a critical threshold estimated to be 0.12 mol m^–3 water. The results also showed that the area which was unfavourable to root growth corresponded to the water table topped with a capillary zone of approximately 6 cm. Once the critical threshold had been reached, it was the water-table duration that explained root behaviour and the first effects were perceptible after approximately 48 h. On the basis of these results, two stress variables were analysed: water-table duration in the root zone (WTD) and proportion of roots in the water table (RPWT). The RPWT variable gave the best results within the two experimental contexts. In the case of the permanent regime, this variable made it possible to consider the root proliferation observations made above the saturated zone. Equations linking the stress variable RPWT to the growth indices are proposed that offer new perspectives to modelling waterlogging effects.     

Comparative study on in vitro stationary culture system or superfusion culture system of cow mammary tissue]

Dairy cow mammary tissue was cultured in superfusion system or stationary system, and the influence of these two methods in the activity and ultrastructure of tissue was investigated according to LDH vigor, trypan blue dying, agrose gel electrophoresis, transmission microscope observation. The results showed that the mammary tissue cultured in superfusion system could keep normal tissue activity and ultrastructure within 12-60 h in DMEM plus 10% calf serum, while mammary tissue stationary culture could keep normal tissue activity and ultrastructure within 60-108 h. Both culture systems had some advantages and disadvantages.     

A flow-through tissue culture system with fast dynamic response

A flow-through system in which monolayer cells, growing on a 50-cm² glass surface, are in contact with a film of medium with a thickness of 0.14 mm, is described. For murine B16 melanoma cells, the loading capacity is 8.10⁶ cells. The flow-through principle permits frequent off-line or on-line detection of medium constituents for a period on the order of days. The system has a fast dynamic response. With off-line radiochemical detection, the system was applied to the uptake of uridine and excretion of uracil over a period of 45 h. With on-line fluorescence detection, the interaction between the cells and two anthracycline analogs was monitored. The cells can be easily observed with a light microscope.     

A multi-station culture force monitor system to study cellular contractility

Cellular contraction contributes to the formation of scar tissue, which is characterized by an over-produced, disorganized collagen matrix. To study the contractility of cells in vitro and its potential contribution to scar tissue formation, we have developed a multi-station culture force monitor (CFM) system. This system consists of four vertical cantilever beams with semiconductor strain gages and a computerized data acquisition unit to monitor contractile forces of the cells in a collagen gel. Calibration showed that this system has a highly linear voltage-force relationship (R(2) > 0.99). Further, to demonstrate the applicability of this system, contractile forces of human skin fibroblasts in a collagen gel were measured. These fibroblasts were found to produce an average force of 0.2 nN/cell, which is consistent with the data in literature. The significant advantage of this CFM system is its ability to test multiple samples simultaneously. Therefore, the system can facilitate statistical design and analysis of experiments to study the effects of growth factors (e.g., TGF-betas) on cellular contraction and their potential role in scar tissue formation.     

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting‐related growth regulators

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone‐free medium was conducted by high‐performance liquid chromatography. The endogenous concentration of auxin indole‐3‐acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root‐forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.     

蕨类植物组织培养研究进展(综述)

蕨类植物组织培养可分为以孢子为外植体的无菌播种和以孢子体为外植体进行的组织培养.本文简要介绍国内外蕨类植物组织培养研究概况,综述培养基成份、培养方式及培养条件对组织培养过程中生长发育的影响,同时介绍蕨类植物组织培养中世代转换的诱导及调控.     

A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.     

A tissue culture system for different germplasms of indica rice

Agrobacterium-mediated transformation of indica rice has been manipulated in only a limited number of cultivars because the majority of indica varieties are recalcitrant to in vitro response. Establishment of a highly efficient and widely used tissue culture system for indica rice will accelerate the application of transformation technology in breeding programs and the study of the functions of indica-specific genes. By manipulating plant growth regulators, organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds representing different indica rice germplasms. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. The results suggest that this optimized tissue culture system will be widely applicable for the tissue culture of indica varieties. Electronic Supplementary Material Supplementary material is available for this article at The first two authors contributed equally to this work.     

Inoculation and tissue distribution in pilot-scale plant root culture bioreactors

Summary Inoculation of large-scale plant root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto process packing elements (e.g. distillation packings), randomly distributed within the reactor, in a bubble column operation. These two techniques have been successfully used to inoculate a 14 L pilot-scale reactor which is subsequently operated as a trickle bed reactor.     

A phage display system designed to detect and study protein-protein interactions.

Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.