Medium Osmotic Potential Research Articles

Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm.

Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied. The conditions at the recovery stage play a vital role in supporting material regrowth after cryopreservation and, when optimized, may shift the life-and-death balance toward a positive outcome. In this contribution, we provide an overview of the five main strategies available at the recovery stage to improve post-cryopreservation survival of in vitro plant materials and their further proliferation and development. In particular, we discuss the modification of the recovery medium composition (iron- and ammonium-free), exogenous additives to cope with oxidative stress and absorb toxic chemicals, and the modulation of medium osmotic potential. Special attention is paid to plant growth regulators used at various steps of the recovery process to induce the desired morphological response in cryopreserved tissues. Given studies on electron transport and energy provision in rewarmed materials, we discuss the effects of light-and-dark conditions and light quality. We hope that this summary provides a helpful guideline and a set of references for choosing the recovery conditions for plant species that have not been cryopreserved. We also propose that step-wise recovery may be most effective for materials sensitive to cryopreservation-induced osmotic and chemical stresses.

Improving cotton embryo culture by simulating in ovulo nutrient and hormone levels

Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton (Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, starting with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L−1 casein hydrolysate plus the following (mmol L−1): d-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8). Mannitol was added to achieve a medium osmotic potential of −1.10 MPa, and an atmospheric O2 tension of 3.3 mol m−3 at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 μmol L−1 indole-3-acetic acid, 80–90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four- to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2- to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote transformation protocols.

Acidiferrobacter thiooxydans, gen. nov. sp. nov.; an acidophilic, thermo-tolerant, facultatively anaerobic iron- and sulfur-oxidizer of the family Ectothiorhodospiraceae

A comprehensive physiological and phylogenetic characterisation was carried out of "Thiobacillus ferrooxidans" m-1, an acidophilic iron-oxidizing bacterium first described over 25 years ago. Phylogenetically, strain m-1 is a gammaproteobacterium, most closely related to alkaliphilic Ectothiorhodospira spp. and only distantly to iron-oxidizing acidithiobacilli. Physiological examination confirmed that strain m-1 can grow autotrophically not only by ferrous iron oxidation but also, in contrast to previous reports, by oxidation of elemental sulfur, sulfide and tetrathionate, using either oxygen or ferric iron as terminal electron acceptor. The bacterium was also found to be thermo-tolerant, growing optimally at 38°C and up to a maximum of 47°C. Growth in liquid media required an external osmotic potential of >2 bar, and was optimal at ~5 bar, though no growth occurred where the medium osmotic potential was close to that of sea water (~26 bar). From this, it was concluded that strain m-1 is a moderate osmophile. Strain m-1 was also shown to be diazotrophic and tolerant of elevated concentrations of many metals typically found in mine-impacted environments. On the basis of these data, m-1 is proposed as the type strain of a new genus and species of bacteria, Acidiferrobacter thiooxydans (DSM 2392, JCM 17358).

Effect of polyethylene glycol (PEG) and sodium chloride on growth, chlorophyll, mineral ions and organic solutes contents, and enzymes activity inPhaseolus vulgarisseedlings

The experiment was made by using different concentrations of polyethylene glycol (PEG) or salt solutions to decrease the osmotic potential of the growth medium to reveal the response of Phaseolus vulgaris to water and salt stresses. No germination (emergence of the radicles) occurred at medium osmotic potential lower than −1.0 MPa in all treatments. It was found that the activity of α-amylase and protease, and contents of proline, saccharides and the soluble proteins decreased in the germinating seeds during 3-day stress. However, after 10-day stress, the contents of organic solutes and the mineral uptake were also significantly reduced under stress. The seedlings under water stress induced by PEG were affected much more than under salinity. This may be due to the maintenance of a higher succulence under salt stress than under PEG-induced water stress.

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture.

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2-7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2-7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.

The effect of osmotic potential on anther culture in spring wheat ( Triticum aestivum )

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to −2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with −0.9 to −1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.

Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by microprojectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclohexylcarbodiimid, indicating that the cytoplasmic DHN1 was newly synthesized under osmotic stress. Furthermore, ABA promoted the presence of green fluorescence in the cytoplasm, and the GFP fluorescence was visualized within a shorter time under a higher osmotic potential.

Sugar Alcohols Display Nonosmotic Roles in Regulating Morphogenesis and Metabolism in Plants that do not Produce Polyols as Primary Photosynthetic Products

Sugar alcohols are frequent supplements to media of cultures from plant species that do not produce polyols as primary photosynthetic products. They comprise one of the major medium constituents and thereby contribute substantially to the medium osmotic potential. It is generally believed that plants that do not have a native pathway for sugar alcohol biosynthesis are also deficient in pathways to assimilate them. Therefore, polyol-media addenda are generally considered to be metabolically inert, and in vitro polyol-dependent phenomena are defined predominantly as mere osmotic (stress) effects. The article shows that this concept does not provide satisfactory explanations for experimental results described in the literature. Sugar alcohols commonly penetrate cells, accumulate, can be translocated and, whenever stringently tested, have always been found to be metabolized to various degrees. Heterotrophic cultures, such as those of tobacco, maize, rice, citrus and chicory, can adjust their metabolism from consumption of saccharides to that of polyols as carbon-energy source when the medium is sucrose-deficient or when exogenous saccharides are depleted to a level lower than a threshold. concentration. In these, as well as in other cultures (i.e. of barley, tobacco, or tomato), sugar alcohols stimulate specific molecular and physiological responses that do not belong to primary carbon metabolism. These responses are dependent on chemical stimuli rather than being mediated by nonspecific, physical, osmotic signals in the medium. One conclusion of this review is that it would be wise to assume that sugar alcohols may be metabolized until shown otherwise. Further, it is suggested that polyols are perceived by cells as chemical signals. This hypothesis is compatible with the emerging conception of sugars (hexoses) as signals in plants, distinctly separate from their possible metabolic and osmotic roles. At very high concentrations provided in vitro , sugar alcohols can probably in some cases be perceived as chemical stress agents, while in other instances they possibly act as protectants against unidentified detrimental medium constituents. In view of the multiple ways of polyol action, administration of these compounds as a means of water stress simulation can lead to misinterpretations of experimental results.

Effect of different salts of sodium and potassium on the growth of Atriplex prostrata (Chenopodiaceae)

Although there are a variety of ions occurring in the soil throughout most of North America, the majority of halophyte literature focuses on the effects of NaCl on plants. In this study, a comparison is made of the effects of NaCl, KC1, Na2SO4, and K2SO4, on growth of the halophyte Atriplex prostrata Boucher ex DC (SYN: A. triangularis Willd.) at 0, ‐0.75, ‐1.00, and ‐1.50 MPa. Plant survival, height, number of leaves, nodes, and branches were recorded weekly. Photosynthesis was measured once before plants were harvested and dry mass was determined after one month. Content of Na+, K+,‐Mg2+, and Cl‐ in plant tissue was also measured. A general trend observed was that all plant growth parameters decreased with a lowering of the medium osmotic potential, and that K+ salts were more inhibitory than Na+ salts. Ion content of plant tissue generally increased with a lowering of osmotic potential. Our data indicated that K+, a plant macronutrient, was more inhibitory to plant growth than Na+. It is possible that halophytes such as Atriplex prostrata could use Na+ as an osmoticum to adjust the vacuolar water potential, but were unable to use K+ for this function because of a specific ion toxicity. The inhibitory effect of salt on plant growth parameters and survival follow the pattern; K2SO4 >KCl>Na2SO4=NaCl.

Effect of water and salt stresses on growth, chlorophyll, mineral ions and organic solutes contents, and enzymes activity in mung bean seedlings

The experiment was made by using different concentrations of polyethylene glycol (PEG) or salt solutions to decrease the osmotic potential of the growth medium to reveal the response of mung bean (Vigna radiata) to water and salt stresses. No germination (emergence of the seedling) occurred at medium osmotic potential lower than -1.0 MPa in all treatments. It was found that the activity of α-amylase and protease, and contents of proline, saccharides and the soluble proteins decreased in the germinating seeds during 3-d stress. However, after 10-d stress, the contents of organic solutes and the activity of the hydrolytic enzymes increased. Growth, chlorophyll content and mineral uptake were also significantly reduced under stress. The seedlings under water stress induced by PEG were affected much more than under salinity. This may be due to the maintenance of a higher succulence under salt stress than under PEG-induced water stress.

Effect of iso-osmotic levels of salts and PEG-6000 on enzymes in germinating pea seeds

Effects of iso-osmotic levels of salts (NaCl, CaCl2, Na2SO4) and PEG-6000 on the activity of hydrolytic and nitrogen assimilatory enzymes in pea embryo axis and coty ledon were studied. The activity of nitrate reductase and nitrite reductase in embryo axis and cotyledon and the activity of protease and α-amylase in cotyledon decreased with decreasing medium osmotic potential as compared to control at all the stages of seedling growth. The activity of protease and amylase increases with increasing levels of stress in embryo axis. Sodium chloride induced, stress had more deleterious effects on the activity of nitrate reductase, nitrite reductase and αamylase followed by other salts and PEG-6000. On the other hand, CaCl2 induced salt stress was more depressive for protease activity. The maximum increase in the activity of protease and amylase was observed in embryo axis at higher concentration of salts and PEG-6000.

Osmotic potential of media affecting green plant percentage in wheat anther culture

The percentage of green plants in anther culture is known to be controlled by the genetics of anther donor materials. The objective of this study was to determine whether components in the culture media also would have a significant influence on the percentage of green plants from wheat anther culture. Anthers of a spring wheat cultivar, "Pavon 76", were cultured on potato 4 (P4) induction media with various modifications. Addition of 200 g/l ficoll to the liquid P4 medium significantly increased the percentage of green plants even though the final yield of green plants per 100 anthers was lower than the liquid medium. A higher concentration of maltose (135 g/l) produced significantly higher percentage of green plants than the medium containing 90 g/l maltose or sucrose. These results demonstrate culture medium effects on albinism, indicating that the percentage of green plants in wheat anther culture can be increased by optimizing medium osmotic potential.

Reduced Osmotic Potential Inhibition of Photosynthesis

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH(4)Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH(4)Cl, and the substrate for phosphoribulokinase to increase in the presence of NH(4)Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH(4)Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.Additions of NH(4)Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

Reduced Osmotic Potential Effects on Photosynthesis

Addition of sorbitol, which facilitated reductions in reaction medium osmotic potential from standard (0.33 molar sorbitol, -10 bars) isotonic conditions to a stress level of 0.67 molar sorbitol (-20 bars), inhibited the photosynthetic capacity of isolated spinach (Spinacia oleracea) chloroplasts. This inhibition, which ranged from 64 to 74% under otherwise standard reaction conditions, was dependent on reaction medium inorganic phosphate concentration, with the phosphate optimum for photosynthesis reduced to 0.05 millimolar at the low osmotic potential stress treatment from a value of 0.25 millimolar under control conditions.Stromal alkalating agents such as NH(4)Cl (0.75 millimolar) and KCl (35 millimolar) were also found to affect the degree of low osmotic potential inhibition of photosynthesis. Both agents doubled the rate of NaHCO(3)-supported O(2) evolution under the stress treatment, while hardly affecting the control rate at optimal concentrations. These agents also reduced the length of the lag phase of photosynthetic O(2) evolution under the stress treatment to a much greater degree. The rate-enhancement effect of these agents under the stress treatment was reversed by sodium acetate, which is known to facilitate stromal acidification.The reaction medium pH optimum for photosynthesis under the stress treatment was higher than under control conditions. In the presence of optimal NH(4)Cl, this shift was no longer evident.Internal pH measurements indicated that the stress treatment caused a 0.43 and 0.24 unit reduction in the stromal and intrathylakoid pH, respectively, under illumination. This osmotically induced acidification was not evident in the dark. The presence of 0.75 millimolar NH(4)Cl partially reversed the osmotically induced reduction in the illuminated stromal pH. It was concluded that stromal acidification is a mediating mechanism of the most severe site of low osmotic potential inhibition of the photosynthetic process.

Responses of Stomata and Water, Osmotic, and Turgor Potentials of Jojoba to Water and Salt Stress1

AbstractJo]oba (Simmondsia chinensis (Link) Schneider) is desert shrub which can provide a much needed substitute for sperm whale oil. Because of the absence of soil‐plant‐water data for jojoba and the limited amount of such information for desert plants in general, selected plants were water‐ and salt‐stressed in greenhouse soil and sand culture experiments. Responses of leaf water, osmotic, and turgor potentials to stress and associated stomatal behavior were studied.Leaf water potentials of well watered jojoba plants (measured with thermocouple psychrometers) averaged —20 bars in the daytime and were generally below —10 bars at night, very low in comparison to most mesophytic plants. The recovery of plants which were water stressed to leaf water potentials below —50 bars (soil water potentials below —40 bars) indicates great drought tolerance. Turgor potential decreased linearly with decreasing leaf water potential, showing a lack of osmotic adjustment as leaf water potentials fall below about —30 bars. Turgot potential reached zero at a leaf water potential of about —35 bars, and dropped as low as —10 bars under the most severe water stress without wilting of mature leaves. Plants in the salinity experiment showed osmotic adjustment down to a root medium osmotic potential of —9 bars.Values of leaf conductance decreased markedly only at very low xylem pressure potentials (pressure chamber measurements) and soil water potentials (—40 bars and —20 bars, respectively), corresponding to about the same values at which turgor potential reached zero. Stomatal closure was continuous with increasing water stress, rather than occurring abruptly at a threshold. There was no significant decrease in leaf conductance at root medium osmotic potentials as low as —9 bars in the salinity experiment.Conductances of well watered plants to water vapor on lower leaf surfaces were typically similar or greater in value than those of upper leaf surfaces, whereas plants under water stress showed significantly higher conductances on their upper leaf surfaces rather than lower leaf surfaces.Leaf xylem pressure potentials decreased with increasing transpiration rate under non‐limiting soil water conditions and decreased below values predicted for well watered plants at the same transpiration rate as soil water potentials decreased. Thus it was possible to make an estimate of edaphic limitations of water absorption based on leaf xylem pressure potentials despite variation in the pressure potentials in response to microclimatic changes.

Timing of Environmental Stress Affects Growth, Water Relations and Salt Tolerance of Pinto Bean

ABSTRACT THE influence of a cool, humid vs. a hot, dry environment on the growth, water relations, and salt tolerance of pinto bean (Phaseolus vulgaris L.) was determined at several salinity concentrations of the root medium. The effects of changing from the cool, humid to the hot, dry environment for 2 wk during the vegetative and the flowering growth stages were also investigated. Growth and pod yield of bean plants exposed to the hot, dry environment during the vegetative stage were comparable with those of plants continuously exposed to the cool, humid environment. Exposure to the hot, dry environment during flowering, however, reduced yields significantly, but not to levels for plants exposed continuously to the hot, dry environment. This inter-action with environment was the same for all salinity levels. Bean grown in the cool, humid environment tolerated higher salt levels than those predicted from published tolerance data. Transpiration rates changed rapidly in response to environmental changes. Differences in transpiration decreased as salinity increased. Linear relationships were found between the osmotic potential of the root medium and leaf water potential and its components. Stomatal conductance was also found to be linearly related to leaf water potential and its components. Stomatal conductance was higher in the cool, humid than in the hot, dry environment for any value of the root medium osmotic potential.