Induction Of Osmotic Stress Research Articles

Impact of Fire Recurrence and Induced Water Stress on Seed Germination and Root Mitotic Cell Cycle of Pinus pinaster Aiton

Climate change will increase the frequency of drought, heat waves, and wildfires. We intended to analyse how fire recurrence and/or induced water stress can affect seed germination and root cell division in Pinus pinaster Aiton. Seeds from stands with no prior fire history and from post-fire regeneration (in areas burnt once, twice, and thrice) in northern Portugal were germinated in distilled water (control) and polyethylene glycol (PEG) to simulate water stress for four weeks, followed by a recovery period. Roots were analysed cytogenetically. The germination index of the Pinus pinaster seeds was not statistically influenced by the induction of osmotic stress, nor by the fire recurrence of the stands. The mean germination time (MGT) was 10–29 days and 1–36 days for the stress and recovery periods, respectively, and increased with PEG concentration. The 20% PEG treatment inhibited root growth after germination. The 10% PEG treatment induced a high frequency of cytogenetic anomalies, mostly in the sites which experienced fire exposure. While fire recurrence did not affect the germination rate, it seemed to reduce the water stress response, negatively impacting cell division and impair root growth.

ФИЗИОЛОГО-БИОХИМИЧЕСКИЕ ХАРАКТЕРИСТИКИ МИКРОПОБЕГОВ ЧАЯ (Camellia sinensis L.) В УСЛОВИЯХ in vitro: НОРМА, ОСМОТИЧЕСКИЙ СТРЕСС, ВЛИЯНИЕ КАЛЬЦИЯ

Stress tolerance is an important trait, that determines the productivity of plants under drought, hypothermia, mineral deficiency, and salinity. Numerous studies of various agricultural crops (J.K. Zhu, 2016; E. Fleta-Soriano, S. Munné-Bosch, 2016), including tea crop (Camellia sinensis L.), were aimed at solving this problem due to the global aridization of the climate. (T.K. Maritim et al., 2015; L.S. Samarina et al., 2019). Along with the sufficiently detailed physiological, biochemical and molecular studies of tea drought tolerance, the exogenous regulation of tolerance by using of chemical and biological substances is still not investigated. In addition, the important role of calcium ions (Ca2+) in the cell recognition of an external stressor by the triggering signal transduction has been shown in many crops (M.C. Kim, 2009; E.G. Rikhvanov et al., 2014). In these studies, tissue culture media supplemented with the osmotically active substances (R.M. Pérez-Clemente et al., 2012; M.K. Rai et al., 2011) and artificial biosystems (microshoots and tissues in vitro), are often used as “drought models” to reveal cellular adaptation mechanisms. However, just a few studies were conducted aimed at deciphering the biochemical and molecular responses of tea plant to stress using tissue culture tool (L.S. Samarina et al., 2018; M.V. Gvasaliya et al., 2019). In this article, for the first time, we investigated the role of calcium in plant adaptation to long-term osmotic stress based on earlier published protocols of tea tissue culture (M.V. Gvasaliya, 2013) and osmotic stress induction protocols. We also demonstrated the prospect of studying the role of exogenous inducers in increasing plant tolerance using “drought models”. This work aimed to identify the effect of different concentrations of calcium (Ca2+) in the culture medium on the functional state of tea microshoots grown under mannitol-induced osmotic stress in vitro comparing with control. The changes in morphophysiological state of the leaves, leaves water content, cells membrane permeability, malondialdehyde, proline, and photosynthetic pigments were analyzed. It was found that increased Ca2+content in the nutrient medium (from 440 to 880 mg/l) resulted the slower leaves development and significant decrease of malondialdehyde and cell membranes permeability of tea microshoots (by 50 %, р ≤ 0.05) during the long-term cultivation of tea microshoots in vitro (4 months), indicating inhibition of lipid peroxidation processes. The addition of mannitol (40 g/l) to the culture medium reduced the water content of the shoots (on average by 2 %, р ≤ 0.05), thereby forming light osmotic stress, which led to the accumulation of proline (an increase of 30-40 %, р ≤ 0.05), as well as to the structural and functional rearrangement of the photosynthetic apparatus (a decrease in the amount of photosynthetic pigments by an average of 35-40 %). In addition, a significant decrease of malondialdehyde (by 50-70 %, p ≤ 0.05) and the intensity of electrolyte leakage from leaf tissues (on average by 50 %, p £ 0.05) were observed, indicating a less pronounced oxidative stress in comparison with control (without mannitol). An increase in the Ca2+ concentration in the nutrient medium (from 440 to 880 mg/l) (in the presence of mannitol) did not significantly affect the water content in the leaves and the photosynthetic apparatus (content and ratio of chlorophylls/carotenoids). An insignificant effect of calcium (in the presence of mannitol) manifested itself in a significant decrease in malondialdehyde by 20 μmol/g dry weight. Consequently, the increased concentration of calcium (660-880 mg/l) in the nutrient medium provides an improvement in the functional state of long-term cultivated tea microshoots in vitro (4 months) by reducing the activity of lipid peroxidation in membranes and increasing their stability. The revealed patterns confirm the positive role of calcium ions in the reduction of combined oxidative stress caused by long-term cultivation of plants in vitro in combination with osmotic stress.

The Calcium-Responsive Phospholipid-Binding BONZAI Proteins Control Global Osmotic Stress Responses in Plants Through Repression of Immune Signaling

SummaryHyperosmotic stress caused by drought and salinity is a significant environmental factor that limits plant growth and agricultural productivity. Plants respond to osmotic stress by activating Ca2+ signaling, accumulating the stress hormone abscisic acid (ABA), reprogramming gene expression and altering growth. Despite intensive efforts, no global regulators of all of these responses have been identified. Here, we show that the Ca2+-responsive phospholipid binding BONZAI proteins are critical for Ca2+ signaling, ABA accumulation, gene expression reprogramming and plant growth under osmotic stress by antagonizing plant immune responses mediated by intracellular immune receptor NLRs. Using a Ca2+-imaging-based forward genetic screen, we found that the Arabidopsis osmo1/bon1 mutant plants display a reduced cytosolic Ca2+ signal in response to hyperosmotic stress. The bon1/bon2/bon3 triple mutants are impaired in osmotic stress induction of gene regulation and ABA accumulation. Importantly, the bon mutants are hypersensitive to osmotic stress inhibition of plant growth. These defects were suppressed by mutations in the immunity regulator SNC1 or PAD4. Our findings suggest that that BON proteins function as global regulators of plant osmotic stress responses by repressing immune signaling.

A MAP kinase‐driven mechanical stress response mediates the hypertrophic response in skeletal muscle

When cells are exposed to unfavorable conditions they cope by activating cellular stress responses. Such signaling depends on activation of the p38 and JNK MAP kinases and is crucial for both general tissue homeostasis and a range of physiological reactions. However, the mechanisms linking diverse stress stimuli to MAP kinase activation remain poorly understood.Here we show how a specific MAP3 kinase mediates activation of p38 and JNK upon hyperosmotic‐ and mechanical stress. Employing fluorescence microscopy techniques, we show that this MAP3 kinase translocates from the cytoplasm to the nuclear lamina and other detergent‐resistant structures in the nucleus upon osmotic stress induction. Here, it co‐localizes with structures rich in DNA damage response factors such as 53BP1, but void of DNA strand breaks, which were shown to constitute nuclear sensor domains for mechanical stress.Mechanical stress is experienced in skeletal muscle due to the repeated contraction of muscle fibers upon physical exercise. The signaling from these events underlie the hypertrophic response, where muscles respond to exercise by growing larger and stronger. We show that the above signaling pathway operates during exercise‐associated stimulus of C2C12 myotubes, where it is responsible for the production of a range of inflammatory transcripts and myokines (such as IL6). Consequently, in a mouse model deficient for the MAP3 kinase, we see clear signs of pathology associated with decreased muscle repair and adaptation to exercise.In conclusion, our work provides novel insight into the mechanisms by which cells sense and react to mechanical stress stimuli and identify skeletal muscle as a major target organ upon deficiency in these responses.

The Use of CRISPR-Cas9 Genome Editing to Determine the Importance of Glycerol Uptake in Wine Yeast During Icewine Fermentation

The high concentration of sugars in Icewine juice causes formidable stress for the fermenting Saccharomyces cerevisiae, causing cells to lose water and shrink in size. Yeast can combat this stress by increasing the internal concentration of glycerol by activating the high osmolarity glycerol response to synthesize glycerol and by actively transporting glycerol into the cell from the environment. The H+/glycerol symporter, Stl1p, has been previously characterized as being glucose repressed and inactivated, despite osmotic stress induction. To further investigate the role of Stl1p in Icewine fermentations, we developed a rapid single plasmid CRISPR-Cas9-based genome editing method to construct a strain of the common Icewine yeast, S. cerevisiae K1-V1116, that lacks STL1. In an Icewine fermentation, the ∆STL1 strain had reduced fermentation performance, and elevated glycerol and acetic acid production compared to the parent. These results demonstrate that glycerol uptake by Stl1p has a significant role during osmotically challenging Icewine fermentations in K1-V1116 despite potential glucose downregulation.

Effect of exogenous stress factors on the biosynthesis of carotenoids and lipids by Rhodotorula yeast strains in media containing agro-industrial waste

In this study, we aimed to determine the effect of exogenous stress factors (sodium chloride as osmotic stressor, hydrogen peroxide as an inducer of oxidative stress, white light irradiation, and low temperature) on the biosynthesis of carotenoids and lipids by red yeast (Rhodotorula glutinis, R. mucilaginosa, and R. gracilis) during cultivation in media containing potato wastewater and glycerol. According to our results, the yeast were able to grow and biosynthesize lipids and carotenoids in the presence of the applied stress factors. Low temperature caused an increase in the biosynthesis of intracellular lipids and carotenoids. R. gracilis synthesized lipids (21.1 g/100 gd.w.) and carotenoids (360.4 µg/gd.w.) in greater quantities than that of other strains. Under these conditions, there was also an increase in the content of unsaturated fatty acids, especially linoleic and linolenic acids. The highest percentage of polyunsaturated fatty acid (PUFA) (30.4%) was synthesized by the R. gracilis yeast after cultivation at 20°C. Their quantity was 2.5-fold greater than that of the biomass grown in control conditions. The contribution of individual carotenoid fractions depended both on the yeast strain and the culture conditions. Induction of osmotic stress and low temperature intensified the biosynthesis of β-carotene (up to 73.9% of the total carotenoid content). In oxidative stress conditions, yeast synthesized torulene (up to 82.2%) more efficiently than under other conditions, whereas white light irradiation increased the production of torularhodin (up to 20.0%).

Development of hyper osmotic resistant CHO host cells for enhanced antibody production

Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328mOsm/kg) or hyper-osmolality (over 450mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process.

Mitigating effects of acetylcholine supply on soybean seed germination under osmotic stress

Acetylcholine (Ach) is a common neurotransmitter in animals, also synthesized in plants, which can have an influence on plant response to stress, also acting as a signaling molecule between root and shoot. The objective of this study was to analyze the possible mitigating effects of exogenous application of Ach on soybean germination under different levels of osmotic potential. The experiments were conducted with soybean [Glycine max (L.) Merrill] genotype Intacta. The seeds were first treated with Ach solutions with the following concentrations: 0.0 (control); 0.5; 1. 0 and 2.0 mM. Then, the seeds were subjected to two water potentials, −0.5 and −1.0 MPa, reached by using mannitol solutions, for the induction of osmotic stress, and a control condition with distilled water. Thus, 12 treatments were established in a double factorial 4 × 3, with 4 levels of Ach and 3 osmotic potential treatments (0.0, −0.5 and −1.0 MPa) with four replicates per treatment. The results showed that the concentration of 1.0 mM Ach, without osmotic stress, presented higher values for total dry mass of the seedlings compared to the control treatment (without Ach supply). In the treatments conducted to test the effectiveness of Ach on the mitigation of severe osmotic stress effects (−1.0 MPa), results showed that the concentration of 0.5 mM Ach showed positive results for the following parameters; dry weight of shoot, root dry weight, total dry mass, which were significantly higher than treatment under 1.0 MPa.

English

In vitro propagation of banana preferably use sword sucker as explant source where microbial contamination poses a great problem in establishment of aseptic cultures. This study demonstrates up to 36% reduced microbial contamination in aseptic culture establishment and subsequent micropropagation due to osmotic stress induction in the banana suckers. Osmotic stress was induced by keeping the freshly collected suckers in shade and measuring fresh weight at 0, 7, 14, 21, and 28 days interval to ascertain loss of moisture. Stress induced for 21 days showed 58.85% moisture loss showing lowest contamination upto 40% against 76% for fresh suckers. Micropropagation of Musa sp. (Malbhog variety) through shoot tip culture of stressed suckers was carried out in Murashige and Skoog (MS) medium supplemented with 1 to 2 mg/l  6-benzyl-aminopurine (BAP), 2 to 4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l  α-naphthalene acetic acid (NAA). Multiplication of plantlets was observed till 6th passage. From 19 aseptically established explants of 21 days stressed sucker sub cultured in MS medium fortified with 1.0 mg/l BAP and 0.5 mg/l NAA, for 6th passage produced 5122 plantlets. Regenerated micro shoots were rooted in MS medium fortified with 0.5 mg/l IAA. The plantlets were hardened in polybag containing soil and seasoned cow dung.   Key words: Osmotic stress, shoot tip culture, in vitro, Musa, Malbhog.

Trace elements’ uptake and antioxidant response to excess of manganese in in vitro cells of sensitive and tolerant wheat

Manganese, a microelement important for plant metabolism, when accumulated at higher doses, may act as a stress factor. Such action of this element is not fully recognized and is currently being intensively studied. The influence of manganese, at high (1, 2 and 3 mM) concentrations, on the induction of oxidative stress in wheat cells was studied under in vitro conditions. Calli of two wheat cultivars, different in terms of stress tolerance, were cultured for 7 days on Murashige–Skoog media with or without auxin (2,4-D) and with additional Mn supplementation. Changes of malondialdehyde (MDA) concentration and the activity of enzymes (SOD, CAT and POX) involved in oxidative response as well as the accumulation of Mn and elements essential for plant development (Mn, Mg, S, Ca, K, P, Na, Zn, Mo, Cu, Fe) were detected. Moreover, proline and carbohydrate contents were determined to check the induction of osmotic stress. An increase of lipid peroxidation (expressed by MDA content), induced by ROS generation, was smaller in the tolerant (‘Parabola’) cultivar than in the sensitive one (‘Raweta’). The activation of antioxidative enzymes was more effective in the cells of tolerant wheat, where a lower quantity of Mn was accumulated. Mn uptake was correlated with a decrease of an amount of almost all the investigated elements. Auxin presence in the culture media accelerated the stressogenic effect of Mn.

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis.

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.

Effect of salinity and PEG-induced water stress on water status, gas exchange, solute accumulation, and leaf growth in Ipomoea pes-caprae

This study was designed to evaluate the response of I. pes-caprae, a pantropical species growing down to the high tide mark on the beach, under low and high drought and saline stresses and under both stresses applied simultaneously. Leaf water relations, gas exchange parameters, and leaf expansion and production rates were evaluated using NaCl and Polyethylene glycol 6000 to adjust the iso-osmotic potential in the nutrient solution to −0.5 and −1.0 MPa. Additionally, both osmotica were combined in the quantities required to allow that both solutes contribute in the same proportion to the total reduction in the solution water potential. The leaf osmotic potential decreased more rapidly at low stress levels and was always lower in plants growing under salinity. At long term, the osmotic adjustment was reached in all treatments, but under high stress, the osmotic adjustment was delayed and plants experimented eventual turgor loss. The simultaneous addition of NaCl and PEG accelerated turgor recovery after the induction of osmotic stress, compared with both agents applied individually. Na + accumulation was enhanced in plants by salt addition, while proline content per leaf water unit increased significantly only at high stress levels. In all treatments maximum assimilation rate and stomatal conductance decreased the first day after addition of salt and/or PEG, and thereafter progressively increased throughout the treatment. At short term and at high stress level, the leaf expansion rate (LER) decreased compared with control plants. When both stresses were applied simultaneously, the effects on LER and leaf area were less detrimental when compared with each stress separately. The results suggest that I. pes-caprae is more susceptible to high water limitations than to soil salinity, while the water and carbon balance were always enhanced when both stresses were applied simultaneously. Thus, although salinity and drought stress are physiologically related and the tolerance mechanisms overlap, some aspect of plant physiology and metabolism may differ when the plant experiments single saline and water or both stresses simultaneously.

Long-term low level glucocorticoid exposure induces persistent repression in chromatin

Environmental exposure to low concentration hormones can have permanent epigenetic effects in animals and humans. The consequence of long-term low concentration glucocorticoid exposure was investigated in cell culture using glucocorticoid responsive genes organized in alternative chromatin structures. The MMTV promoter is induced by short-term glucocorticoid exposure on either an integrated (normal chromatin) or transient (unstructured chromatin) promoter. Longer hormone treatment causes a transient refractory repression of only the integrated promoter. Exposure to low concentrations of hormone for several passages persistently represses the integrated MMTV and endogenous glucocorticoid responsive promoters. The glucocorticoid receptor cannot bind to persistently repressed promoters. Induction by androgens is also inhibited on the repressed MMTV promoter. Similarly, osmotic stress induction of the endogenous Sgk gene is repressed. Persistent repression by glucocorticoids targets glucocorticoid responsive genes using a chromatin-dependent mechanism that disrupts binding of both GR-dependent and GR-independent transcription complexes.

Glycogen synthase kinase-3β and p38 phosphorylate cyclin D2 on Thr280 to trigger its ubiquitin/proteasome-dependent degradation in hematopoietic cells

Cyclin D2 plays an important role in regulation of hematopoietic cell proliferation by cytokines and is implicated in oncogenesis of various hematopoietic malignancies. However, mechanisms regulating cyclin D2 stability and its expression level have remained to be known. Here, we demonstrate that interleukin-3 signaling stabilizes cyclin D2 by inhibition of glycogen synthase kinase-3beta (GSK3beta) through Janus kinase2-dependent activation of phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway in hematopoietic 32Dcl3 cells. On the other hand, osmotic stress was shown to induce a rapid proteasomal degradation of cyclin D2, which was mediated by activation of p38. GSK3beta and p38 was demonstrated to phosphorylate cyclin D2 on Thr280 in vitro, while a cyclin D2 mutant with this residue substituted with Ala was found to be resistant to ubiquitination and proteasome-dependent degradation in 32Dcl3 cells. Inhibition of the PI3K pathway or induction of osmotic stress also caused a rapid proteasomal degradation of cyclin D2 in primary leukemic or myeloma cells. These results indicate that cyclin D2 expression in normal and malignant hematopoietic cells is regulated by ubiquitin/proteasome-dependent degradation that is triggered by Thr280 phosphorylation by GSK3beta or p38, which is induced by inhibition of the PI3K pathway or by osmotic stress, respectively.

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

Separation of Sub-Micron Particles from Soils and Sediments without Mechanical Disturbance

AbstractA method is described for the separation of the finest particles from soils and sediments without mechanical disturbance. Particles are separated through the induction of osmotic stress. Generally, samples are treated with a concentrated sodium salt solution and then exposed to water by diffusion. Naturally sodic samples are simply exposed to water. Solid samples and the swollen and dispersed material they produce are confined by dialysis tubing. Examples show that the method gives a size gradient of particles in a vertical column of suspension. The compositions of particles can vary with size. The method can be used to show the effects on separated particles of ions other than Na+ and also of other physicochemical treatments of soils and sediments. It is inexpensive and requires little labor.

The Growth of Carrot Callus Cultures at Various Concentrations and Composition of Saline Water

Summary Diploid callus cultures of carrots ( Daucus carota ssp. sativa ) were grown on media which contained various concentrations and compositions of salts. Salt stresses were induced by adding various concentrations of the following salt(s) to the media: sea water, synthetic sea water, NaCl, KCI, MgCl 2 , Na 2 SO 4 , H 3 BO 3 . In addition, Mannitol was used in some experiments for the induction of osmotic stress. The salinity stress inhibited the growth of the calli, prevented the formation of green color in the tissue, and caused necrosis and death. We have concluded from these experiments that growth inhibition was caused mainly by the increased osmotic pressure while the inhibition of green color formation and the necrosis were caused mainly by the high concentration of salt(s). Potassium was more toxic to the calli than sodium. The results are discussed in terms of selection systems for the isolation of salinity resistant mutants.