Experimental and Analytical Studies of Pteridophytes: XXI. Investigations on Marsilea

An account is given of the effects of various concentrationsof glucose, sucrose, and fructose on the development and morphologyof aseptic cultures of Marsilea sporelings. The response ofthe sporelings to a range of different sugars and to growthin liquid or on solid media is also described. The results are discussed with special reference to the relationshipbetween nutrition and heteroblastic leaf development. Otheraspects of the influence of various sugars on the growth ofplants in aseptic culture are considered in relation to theexisting literature.     

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmoticstress, created by high agarose or carbohydrate concentrations,on the regeneration of fertile plants from protoplast-denvedcolonies of several indica (IR43, Jaya, Pusa Basmati 1) andjaponica (Taipei 309) rice varieties. Observations of the culturesdeveloped on media containing one of these carbohydrates (cellobiose,fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose),each at 88 mM, indicated that maltose was the preferential carbonsource for the proliferation of embryogenic callus and shootregeneration. Maltose-containing medium induced shoot formationin 24–66% of the protoplast-derived tissues, dependingupon the rice variety, compared to shoot regeneration from 4–32%of the tissues in sucrose-supplemented medium. Media containing288 mM maltose or an equimolar combination of 88 mM maltoseand 200 mM mannitol, caused water loss from calli and promotedthe growth of embryogenic calli. These calli formed shoots withgreater frequencies when subsequently transferred to shoot regenerationmedium with 88 mM maltose. A medium containing 88 mM maltoseand semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarosehad a similar beneficial effect on the growth of embryogeniccalli and simultaneously supported high-frequency (48–55%)shoot formation. The optimum shoot regeneration frequencies(60–78%) were obtained when protoplast-derived colonieswere serially cultured on to shoot regeneration medium containing1.0% (w/v) agarose for 4 weeks, followed by a 2-week cultureperiod on the same medium with 0.5% (w/v) agarose. Plants regeneratedon medium containing maltose and/or 1.0% (w/v) agarose werephenotypically normal and fertile. Key words: Carbohydrates, Oryza sativa L, indica and japonica rice, osmotic stress, plant regeneration, protoplast-derived colonies     

Investigations into the Role of Sucrose in Potato cv. Estima Microtuber Production in vitro

Sucrose has been the carbohydrate traditionally used for potatomicrotuber production. Added to nutrient media, sucrose canact solely as a carbon source, or as an osmoticum, or both.Preliminary tests showed that the osmolarity of sucrose solutionswas increased by autoclaving, indicating some breakdown of thesugar. This was taken into consideration in experiments whichinvolved supplementing 4% sucrose media with sucrose, maltose,glucose or fructose, while keeping the osmotic potential ofthe media constant. A medium concentration of about 400 mM withonly sucrose was more suitable for microtuber production thanmedia supplemented with maltose, glucose or fructose. However,a better microtuber yield was obtained when hexoses were addedthan with unsupplemented 4% sucrose media. When glucose wassupplied at concentrations which had the same number of carbonatoms as 8% sucrose, the high osmolarity inhibited microtuberisation.Sugar movement in the tubering plantlet was followed using radio-labelledsucrose, glucose and fructose. The sucrose was translocatedand used at a faster rate than the other sugars, which tendedto remain in the roots of the plantlets. Furthermore, therewas no difference in microtuber production on media to whichthe sucrose was added before or after autoclaving, indicatingthat levels of breakdown were not severe enough to affect theprocess. Therefore, it is concluded that sucrose acts primarilyas a suitable carbon source for uptake and utilization by theplantlets, but, at 8%, it also provides a favourable osmolarityfor the development of microtubers.Copyright 1995, 1999 AcademicPress Solanum tuberosum (L.), potato, microtuber, media, sugar, sucrose, osmolarity, pH     

Effects of “media osmotic agents” on the growth and morphogenesis ofActinidia deliciosa cv “hayward” callus

Summary The influence of various osmotic agents (carbohydrates) on the morphogenesis and growth of callus ofActinidia deliciosa cv Hayward was studied. Sucrose supported the highest level of growth and the lowest was supported by the sugar alcohols used in the experiments (glycerol, mannitol, sorbitol). The growth and survival of callus were evaluated with different osmotic sources in media containing glycerol, mannitol, or sorbitol at a concentration of 0.2M each for an extended period of eight subcultures (360 days). Two crucial points were identified: until the third subculture (135 days) the vitality seemed to be elevated; whereas the fifth (225 days) seemed to be a “point of no return” for tissues grown in glycerol and mannitol. Pretreatment with osmotic carbohydrates was shown to increase the magnitude of the morphogenetic events of callus subsequently transferred to sucrose-containing medium. Callus grown in the presence of mannitol and sorbitol showed a similar frequency of morphogenetic response. With respect to the media containing glycerol and sucrose, these induced more intense regeneration of shoots. When glycerol was present in the medium, however, we observed a synchronization of the morphogenetic response. Our results suggest that it is possible both to stimulate and to synchronize morphogenesis utilizing osmotic conditioning subcultures.     

Sucrose uptake by the phloem parenchyma of carrot storage root

The characteristics of sucrose uptake into the symplast of phloemtissue discs harvested from fresh, actively-growing carrot storageroots are described. Sucrose uptake exhibited a curvilinearresponse with increasing sucrose concentration. The inhibitorsp-chloromercuribenzenesulphonic acid (PCMBS) and carbonyl cyanidem-chlorophenylhydrazone (CCCP) decreased uptake and resultedin solely linear relationships between uptake and sucrose concentration.These results suggest that active carrier-mediated transportoccurs at the plasmalemma in addition to a diffusive mechanism.The former saturates at a lower concentration (approximately20 mM) than the latter which does not saturate below 100 mM.Though similar in their effect on the ethanol-soluble fraction,CCCP and PCMBS had different effects on the conversion of sucroseto ethanol-insoluble material. Varying the osmotic environment with different mannitol concentrationsdid not affect uptake between 0 and 400 mM mannitol, but didcause an increase at 600 mM mannitol: an effect which may havebeen an artefact of plasmolysis. Metabolic conversion to ethanol-insolubleforms remained unchanged from 0 to 250 mM mannitol and declinedabove this. Thus metabolism, but not uptake may be responsiveto changes in turgor. Key words: carrot, sucrose, uptake, transport, turgor     

Experimental and Analytical Studies of Pteridophytes XXIV. Investigations on Marsilea: 4. ANATOMICAL EFFECTS OF CHANGES IN SUGAR CONCENTRATION

Sporelings of Marsilea, grown aseptically in various concentrationsof sugars, particularly glucose, have been investigated anatomically. It is shown that, corresponding to the features of the externalmorphology, the anatomy of the sporelings from the lower sugarconcentrations (1 and 2 per cent. glucose) has many characteristicsof the water forms of amphibious plants, while that of the sporelingsfrom the higher sugar concentrations (4 and 5 per cent, glucose)has the characteristics of typical land forms. A number of the differences between the two types of sporelingmay be referred to a change in the duration and direction ofcell-division in the rhizome-apex. Views as to the factors influencingthe origin of land or water forms are briefly discussed.     

Water and Solute Transport by Exuding Root Systems of Ricinus communis

The influence of range of concentrations of potassium nitrate(C_m)in the medium surrounding the roots of decapitated Ricinuscommunis plants on: (a) the potassium concentration of the exudate(C_s), (b)the rate of exudation (fH₂O), and (c) the flux of potassiuminto the exudate (f_k) was investigated. A change in C_m couldinfluence exudation by altering the osmotic potential of themedium as will as by a change in potassium concentration, andthese two effect were separated in the following way. the effectof varying the potassium concentration was investigated usingmedia made isotonic with mannitol, whilst the osmotic effectwas investigated using mannitol solutions of various concentrationsbut of equal potassium concentration. An analysis of the resultsrevealed the existence of two salt-transfer processes, one independentof the water flux but dependent on the concentration of potassiumin the medium, the other linearly related to the flux of waterthrough the root system. The implications of these findingsin relation to the intact transpiring plant are discussed.     

Osmotic Stress as a Pregrowth Procedure for Cryopreservation 2. Water Relations and Metabolic State of Sycamore and Soybean Cell Suspensions

Addition of 6 per cent mannitol or sorbitol to liquid culturemedium decreased the water potential (_w) by –0.93 MPa( 382 ± 7 mOsm kg^–1 water). Sycamore cells grownto exponential phase in such media exhibited increased levelsof total and soluble protein and respiratory activity, but decreasedamounts of free proline. Soybean cells showed increased respiratoryactivity and free proline levels, but total protein levels remainedunaffected. Soluble protein levels were reduced under sorbitol-inducedstress. In both species osmotic stress had little effect oncell dry weight. Water relation studies indicate that sycamore cells are capableof much greater osmotic adjustment than soybean cells, and thatmannitol uptake does not contribute significantly to that adjustment. Acer pseudoplatanus L., sycamore, Glycine max L. var. Biloxi, soybean, suspension culture cells, osmotic stress, water relations, metabolism     

Sugar Efflux from Attached Seed Coats of Glycine max (L.) Men.

Ellis, E. C. and Spanswick, R. M. 1987. Sugar efflux from attachedseed coats of Glycine max (L.) Merr.—J. exp. Bot. 38:1470—1483. Sugar efflux (sucrose + glucose) from attached seed coats ofGlycine max (L.) Merr. was measured at high sampling rates toimprove the kinetic characterization of seed coat exudation.This study confirms that sugar efflux in seed coats has at leasttwo components, and demonstrates that the concentration of mannitolosmoticum bathing the seed coat may influence one or both ofthese components. High leaf irradiance increased sugar effluxrelative to a low leaf irradiance at the same mannitol concentration.A high concentration of mannitol (500 mol m³) enhanced sugarefflux relative to a medium concentration (100 mol m³) underboth high and low leaf irradiance. A low mannitol concentration(10 mol m³) stimulated sugar efflux (relative to 100 mol m³)to a greater extent when leaf irradiance was high. Rapid changesin mannitol concentration produced immediate stimulations ofsugar efflux. Effects of osmoticum on sugar efflux are explainedby simultaneous turgor-mediated effects on import of sucroseby the phloem and retrieval of apoplastic sucrose, presumablyby seed coat parenchyma.     

Glycerol production by two filamentous fungi grown at different ionic and nonionic osmotics and cheese whey

Glycerol production by a highly glycerol-producing local isolate (Eurotium amstelodami) and a standard reference isolate (Aspergillus wentii) was markedly enhanced by high saline media. Glycerol concentration depended on the external osmotic. Thus, the highest glycerol concentration was found in the presence of NaCl, followed by KCl, with considerably lower values for MgCl₂ and CaCl₂ saline media. With glucose (5–50%) used as a nonionic osmotic, low levels of glycerol were obtained and the main pool of polyols was mannitol. Glycerol production was gradually increased with the increase of NaCl concentration of cheese whey, reaching maxima by both organisms when whey was supplemented with 8% NaCl (total of 16% NaCl). The quantity of glycerol produced byA. wentii was twice higher than that obtained byE. amstelodami on whey treated with 8% NaCl.     

Effects of external osmolality on polyamine metabolism in HeLa cells

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation.A sudden increase in NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Effect on Transpiration and Water Uptake by Rapid Changes in the Osmotic Potential of the Nutrient Solution

The sudden changes in the rates of transpiration and water uptake which occurred when the osmotic potential of the nutrient solution surrounding the roots of young wheat plants was rapidly changed were studied. The transpiration was measured by the aid of the microwave hygrometer and the water uptake by a recording poto-meter specially built for this investigation. When the osmotic potential of the nutrient solution was rapidly increased by adding mannitol, there was a temporary transpiration increase. The maximum increase was greater but the total time of the temporary increase shorter when a higher mannitol concentration was used. The quantity of water transpired by the shoots due to the temporary transpiration increase seemed to be fairly constant irrespectively of the mannitol concentration. The water transport to the shoots was immediately reduced when the osmotic potential was rapidly increased. The immediate reduction was greater when a higher mannitol concentration was used. After the immediate reduction the rate of water transport increased without delay. When the osmotic potential of the nutrient solution was rapidly decreased by withdrawing mannitol there was a temporary transpiration decrease, and the water transport to the shoots was immediately increased. After this increase the rate of water transport started to decrease at once. When, however, the mannitol concentration had been 0.30 M or higher, the transpiration rate increased progressively, and the change of the rate of water transport was small. The results indicate that the primary effect of the rapidly changed osmotic potential is localized to the root surface. The rapidly reduced water transport to the shoots after adding mannitol brings about the temporary transpiration increase. The course of events after withdrawing mannitol is just the reverse to that when adding mannitol.     

Chronotropic response to hyperosmotic solutions of isolated rat atria

The changes in spontaneous rate of isolated rat atria in response to increased extracellular osmotic pressure were examined using sucrose, urea and several polyhydroxyalcohols (mannitol, glycerol and ethylene glycol) as test solutes. Sucrose, mannitol and urea induced a fall in atrial rate, which was transient with the last compound. On the other hand, media made hyperosomotic by addition of glycerol or ethylene glycol increased the beating frequency. Sucrose effect was not affected by low extracellular calcium, nifedipine or atropine. Glycerol-induced increase in atrial rate was a calcium-dependent mechanism sensitive to nifedipine. Thus, positive chronotropic effect occurs in the rat atria only with certain diffusible solutes which probably promote calcium entry. The response to pure osmotic change, resulting from changes in concentration of ions within the cell as water moves out, is a negative chronotropic effect.     

Uptake of mannitol from the media by in vitro grown plants

Higher plants grown in vitro are very seldom fully autotrophic. Therefore, such cultures are usually supplied with exogenous sugars. However, at higher sugar concentration a decrease in dry matter accumulation is observed which can be explained by a decrease in osmotic potential of the medium.To test this explanation a series of experiments with mannitol, a sugar alcohol often used for simulation of osmotic stress, were performed with excised wheat embryos, rape seedlings and potato stem segments grown in vitro. As the presence of mannitol in the medium caused a significant decrease in dry matter accumulation, the content of mannitol in the shoot tissues was determined using HPLC analysis to estimate the uptake and transport of mannitol from roots to shoots. Mannitol contents up to 30% of dry weight in wheat and 20% in rape and potato shoots were found, indicating that mannitol is easily taken up by in vitro plants and transported to shoots. There were no large changes in the content of glucose, fructose and sucrose caused by the presence of mannitol in the tissues. These data show that mannitol can not be used as an inert osmoticum in in vitro studies.     

Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules

Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In fruits containing 4–6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 m M ), medium (350, 400 or 500 m M ) or high (0.7 or 1 M ) concentration of sucrose and/or mannitol. In pulse-labelling experiments with sucrose and α-aminoisobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules.
In our experiments, 350 m M appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmolarity of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in the seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the "sink strength" of developing seeds is turgor-sensitive.     

Osmotic Stress as a Pregrowth Procedure for Cryopreservation: 1. Growth and Ultrastructure of Sycamore and Soybean Cell Suspensions

Sycamore and soybean cell suspensions were subjected to osmoticstress by culturing for one passage in media supplemented with6 per cent mannitol or sorbitol. The effects on growth wereto reduce cell number and biomass (d. wt) production throughoutthe culture period by about 30 per cent. Ultrastructural studiesat the early exponential phase of culture growth indicated similarreductions in cell wall thickness in both species. Osmoticallystressed sycamore cells became less vacuolate, but no such changeoccurred in stressed soybean cells. Acer pseudoplatanus L., sycamore, Glycine max L. var. Biloxi, soybean, suspension culture cells, osmotic stress, growth, ultrastructure     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Effect of the Osmotic Environment on Assimilate Transport in Isolated Developing Embryos of Maize (Zea mays)

Embryos obtained from developing kernels of maize (Zea maysL.) were incubated in a bathing medium, to measure the effectof the osmotic environment on the balance between uptake andrelease of assimilates by the embryo. Net efflux of sucroseand amino acids from the embryo decreased with increasing mannitolconcentration in the bathing medium and net uptake of [¹⁴C]valine increased with increasing mannitol concentration. Therole of a high osmolality of the seed apoplast in seed developmentis discussed Zea mays, maize, embryo, seed development, assimilate transport, turgor-sensitive transport     

The effect of osmotics on plant cells: a study using 1H NMR relaxation and self-diffusion of water.

The osmotic changes in root cells of Zea mays L. under the effect of mannitol (concentration range 1-15%; range of osmotic pressures: -0.13 to -2.01 MPa) were studied by measuring time and concentration dependence of water self-diffusion constant (Deff) and proton spin-lattice relaxation time (T1) using proton NMR relaxation spectroscopy. In addition, in vivo uptake of Mn2+ by roots after their incubation in mannitol solutions were studied. At low (less than or equal to 5%) and medium (5-10%) concentrations of the osmoticum dehydration takes place proportional to the concentration, whereas membrane destruction occurs at higher concentrations (10-15%). It seems that there is a distribution of cells within root tissue regarding their sensitivity to osmotic stress.     

Maturation of black spruce somatic embryos: Sucrose hydrolysis and resulting osmotic pressure of the medium

The physiological and osmotic roles of sucrose during black spruce (Picea mariana (Mill.) B.S.P.) embryo maturation were investigated. The results showed that when both sucrose and mannitol were present in the medium, the optimum sucrose concentration varied between 4% and 6%. From these data, mannitol does not apparently replace sucrose during the maturation of somatic embryos and therefore it might not be a suitable osmoticum. For the media supplemented with 4% to 12% sucrose and various concentrations of mannitol, the osmotic pressure of the medium rose during maturation, particularly for the highest sucrose concentrations (7% to 12%). Medium containing 3% each of fructose and glucose produced fewer mature embryos compared to the medium with 6% sucrose. An increment in the osmotic potential was observed in medium with 6% sucrose in contrast to that containing 3% each of fructose and glucose. Sugar analysis revealed that the sucrose hydrolysis in the medium was detectable within 1 week of incubation and continued throughout the maturation period. Moreover, no significant uptake of the sugars was detected, since the total amount of fructose, glucose and sucrose remained constant. Our results indicate that the action of sucrose on embryo maturation is mostly achieved through an osmotic control.