Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules

Earthworm egg capsules of five species were compared with regard to survival and water relations upon exposure to controlled dehydration at 20°C. Cocoons of the investigated species all contained about 3.5 g water·g^-1 dry weight when fully hydrated. Approximately 18% of this does not readily freeze upon cooling to -40°C and is referred to as osmotically inactive water. Cocoons exposed to desiccation lose a large proportion of the osmotically active water over 1–4 days until water in the cocoon fluids has equilibrated with surrounding water vapour. The amount of osmotically inactive water, on the other hand, is only reduced by 10–20%. Dendrobaena octaedra was the species most tolerant to drought, its tolerance limit coinciding with loss of practically all osmotically active water. For the five species investigated, there seemed not to be any clear correlation between drought tolerance and microhabitat. Previous investigations have suggested a very close relation between tolerance to dehydration and to subzero temperatures in overwintering earthworm cocoons. Survival at a given level of dehydration at room temperature is less than at temperatures below 0°C, and the tolerance of room temperature dehydration is not closely correlated with cold hardiness across the range of the species studied.Abbreviations dw dry weight - DSC differential scanning calorimetry - fw _pd fresh weight of partially dehydrated cocoons - OAW osmotically active water - OIW osmotically inactive water - Osm osmolality - water potential - R universal gas constant - T absolute temperature - V specific volume of water     

Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis

The loss during desiccation of osmotically active water (OAW), which freezes during cooling to -45 degrees C, and osmotically inactive water (OIW), which remains unfrozen, from the cysts of the potato cyst nematode, Globodera rostochiensis, was determined using differential scanning calorimetry. Exotherms and endotherms associated with non-egg compartments were not detected after 5 min desiccation at 50% relative humidity and 20 degrees C. The pattern of water loss from the cysts indicates that water is lost from compartments outside the eggs first, that nearly all the non-egg water is OAW and that the OIW content of the cyst is contained within the eggs. Water is lost from the eggs only after the OAW content outside the eggs falls below that within the eggs. Both OAW and OIW are lost from the eggs during desiccation but the eggs retain a small amount of OIW. Other animals which survive some desiccation but which are not anhydrobiotic will tolerate the loss of OAW but not the loss of their OIW. Anhydrobiotic animals can survive the loss of both their OAW and a substantial proportion of their OIW.     

Preservation of chitinolytic Pantoae agglomerans in a viable form by cellular dried alginate-based carriers

Improved viability of Gram-negative bacteria during freeze-dehydration, storage, and soil inoculation is of crucial importance to their efficient application. The chitinolytic Pantoae (Enterobacter) agglomerans strain IC1270, a potential biocontrol agent of soil-borne plant-pathogenic fungi, was used as a model organism to study the efficacy of freeze-dried alginate-based beads (macrocapsules) as possible carriers for immobilized Gram-negative bacterial cells. These macrocapsules were produced by freeze-dehydration of alginate gel spherical beads, in which different amounts of bacteria, glycerol, and colloidal chitin were entrapped. Subsequent drying produced different unexpected structures, pore-size distributions, and changes in the outer and inner appearance of the resultant dried cellular solid. With increasing glycerol content, the proportion of larger pores increased. These structures can be related to changes in the slow-release properties of the dried beads. The amount of glycerol in the beads differed from that in the alginate solution as a result of leakage during the beads' preparation and dehydration. Entrapping 10(9) cells per bead produced from alginate solution containing 30% glycerol and 1% chitin resulted in improved (in comparison to other studies) survival prospects (95%) during freeze-drying. Moreover, immobilization of the bacterium sharply improved its survival in nonsterile irrigated and dry soils compared to bacteria in a water suspension. The results suggest that optimized conservation of Gram-negative bacteria in dry glycerol-containing alginate-based cellular solids is not only possible but applicable for a variety of uses.     

玉米胚发育过程中脱水耐性的变化

对离体玉米胚脱水耐性的变化以及不同脱水速率对其脱水耐性的影响进行了研究。授粉后16d的玉米胚能耐轻微脱水,含水量从1.45降低到0.28gH2Og-1DW时胚的萌发率为100%,但含水量低于0.1gH2Og-1DW时胚死亡。胚的脱水耐性随着发育逐渐加强,表现为电解质渗漏速率逐渐降低,萌发率和幼苗干重逐渐增加。授粉后20d胚内超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性较高,过氧化氢酶(CAT)活性较低;授粉后24d,这些酶的活性与授粉后20d的正好相反。脂质过氧化产物丙二醛(MDA)在种子发育过程中呈下降趋势。不同脱水速率明显地影响胚的脱水耐性:在慢速脱水到含水量0.1~0.18gH2Og-1DW时,胚的萌发率和幼苗干重比快速脱水高,电解质渗漏速率比快速脱水低;在快速脱水条件下胚中的SOD、APX活性和MDA含量也比慢速脱水高;CAT活性的变化不明显。     

Pseudomonas fluorescens 134 as a biological control agent (BCA) model in cell immobilization technology

Antifungal activity against Rhizoctonia solani was achieved in vivo through the application of Pseudomonas fluorescens strain 134 encapsulated in sodium alginate beads of different sizes (0.5, 1, and 2 mm). The activity was compared to that obtainable with chemical treatments and bead-derived liquid formulations. The latter was obtained by dissolving alginate beads of 1 and 0.5 mm in 1% Na-citrate solution before application, without any significant (P < 0.05) reduction of bacterial numbers during the dissolution process. The dry bead formulations were applied next to the seeds in plant inoculation experiments, resulting in a reduction of disease symptoms, which were markedly reduced when the liquid formulation was applied. Moreover, the rate of disease symptoms related to liquid formulations from both 1 and 0.5 mm beads was comparable (near to 10%) to that of chemical treatment. Pseudomonas fluorescens strain 134 delivered as both dry and liquid formulations was able to colonize cotton root at a population density of about 10(8) CFU/g fresh root, 15 days after sowing.     

Enhancing carrot somatic embryos survival during slow dehydration, by encapsulation and control of dehydration

In order to obtain dry artificial seeds, carrot somatic embryos were encapsulated and dehydrated. Encapsulation in some hydrogels delayed the dehydration and preserved the water content of carrot somatic embryos. In particular, a matrix made of alginate with gellan gum was found to be the most efficient in maintaining a high water activity (a_w) around somatic embryos. By delaying dehydration, and also rehydration, encapsulation seemed to protect somatic embryos against desiccation and imbibition damages, giving better germination and emergence of cotyledons. Matrices made of alginate mixed with kaolin or gellan gum were particularly adapted to protect the embryos during the dehydration. Apart from the matrix composition, the control of dehydration speed enhanced the survival and regeneration of encapsulated-dehydrated somatic embryos. Using a slow dehydration protocol (95-15% RH—relative humidity into the chamber—in 11.5 days), it was possible to exert different dehydration speeds. Slowing the dehydration between 70 and 45% RH stabilized the water activity (a_w) of the encapsulation matrix, and enhanced the survival and regeneration frequencies of encapsulated-dehydrated embryos. In the absence of any maturing pretreatment, alginate-gellan gum encapsulated carrot somatic embryos, dehydrated to 15% RH, and rehydrated in moistured air (90% RH), germinated up to 72.9%. Therefore, encapsulation in alginate-gellan gum, combined with a slow dehydration, leads to enhance the somatic embryos' desiccation tolerance.     

Dehydration of earthworm cocoons exposed to cold: a novel cold hardiness mechanism

Mechanisms involved in cold hardiness of cocoons of the lumbricid earthworm Dendrobaena octaedra were elucidated by osmometric and calorimetric studies of water relations in cocoons exposed to subzero temperatures. Fully hydrated cocoons contained ca. 3 g water · g dry weight^-1; about 15% of this water (0.5 g·g dry weight^-1) was osmotically inactive or bound. The melting point of the cocoon fluids in fully hydrated cocoons was-0.20°C. Exposure to frozen surroundings initially resulted in supercooling of the cocoons dehydrated (as a result of the vapour pressure difference at a given temperature between supercooled water and ice) to an extent where the vapour pressure of water in the body fluids was in equilibrium with the surrounding ice. This resulted in a profound dehydration of the cocoons, even at mild freezing exposures, and a concomitant slight reduction in the amount of osmotically inactive water. At temperatures around-8°C, which cocoons readily survive, almost all (>97%) osmotically active water had been withdrawn from the cocoons. It is suggested that cold injuries in D. octaedra cocoons observed at still lower temperatures may be related to the degree of dehydration, and possibly to the loss of all osmotically active water. The study indicates that ice formation in the tissues is prevented by equilibrating the body fluid melting point with the exposure temperature. This winter survival mechanism does not conform with the freeze tolerance/freeze avoidance classification generally applied to cold-hardy poikilotherms. Implications of this cold hardiness mechanism for other semi-terrestrial invertebrates are discussed.Abbreviations DSC differential scanning calorimetry - dw dry weight - MP melting point(s) - II water potential - R universal gas constant - T absolute temperature - V specific volume of water     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Partial desiccation induced by sub-zero temperatures as a component of the survival strategy of the Arctic collembolan Onychiurus arcticus (Tullberg)

The mechanism by which the freeze susceptible Arctic collembolan Onychiurus arcticus survives winter temperatures of -25 degrees C in the field is not fully understood but exposure to sub-zero temperatures (e.g. -2.5 degrees C) is known to induce dehydration and lower the supercooling point (SCP). In this study, changes in the water status and certain biochemical parameters (measured in individual Collembola) during a 3-week exposure to decreasing temperatures from 0 to -5.5 degrees C were studied. Osmotically active and inactive body water contents were measured by differential scanning calorimetry (DSC), water soluble carbohydrates by high performances liquid chromatography (HPLC) and glycogen by enzymatic assays. The activity of trehalase and trehalose 6-phosphate synthase were also measured. During the experiment, total water content decreased from 70 to 40% of fresh weight, mostly by the loss of osmotically active water with only a small reduction in the osmotically inactive component. The SCP decreased from -7 to -17 degrees C. Analysis of the results shows that if O. arcticus is exposed to -7 degrees C in the presence of ice, all osmotically active water would be lost due to the vapour pressure gradient between the animals supercooled body fluids and the ice. Under these conditions the estimated SCP would reach a minimum of c. -27 degrees C, but the Collembola may never freeze as all the osmotically active water has been lost, the animal becoming almost anhydrobiotic. Trehalose concentration increased from 0.9 to 94.7&mgr;g mg(-1)fw while glycogen reserves declined from 160 to 7.7 nmol glucose equivalents mg(-1) protein. Trehalase activity declined as the temperature was reduced, while trehalose 6-phosphate activity peaked at 0 degrees C. By adopting a strategy of near anhydrobiosis induced by sub-zero temperatures, O. arcticus, which was previously thought to be poorly adapted to survive severe winter temperatures, is able to colonise high Arctic habitats.     

The state of water in acclimating vegetative buds from Malus and Amelanchier and its relationship to winter hardiness

The relationship between freezable water and cold hardiness during acclimation was studied using vegetative buds from several apple ( Malus domestica Borkh) cultivars and from one saskatoonberry ( Amelanchier alnifolia Nutt. cv. Smoky) cultivar. According to leakage data and visual assessments of cortical browning, vegetative buds of all cultivars were most tolerant to subfreezing temperatures in January. The hardy condition was also associated with maximum tolerance to desiccation. Qualitative features of freezing exotherms (number of peaks and temperature of the transition) were not correlated with the hardy condition in the tissues. However, the amount of unfrozen water, determined by quantifying the energy of the exotherms, increased with increasing hardiness. In buds that survived exposure to −45°C, freezing reduced the intracellular water content, but only to levels above the critical moisture content for desiccation damage. In buds that did not survive exposure to −45°C, freezing reduced the water content to levels equal to or less than the critical moisture content for desiccation damage. These observations suggest that the freezing of water in nonhardy tissue dried the tissue to moisture levels at which severe dehydration damage occurred. It appears that acclimation of vegetative apple buds involves at least two processes: (1) an increase in tolerance to dehydration and (2) an increase in the level of unfreezable water.     

Water Content, Raffinose, and Dehydrins in the Induction of Desiccation Tolerance in Immature Wheat Embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.     

Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.     

Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Accumulation of soluble sugars, heat-stable proteins and dehydrins in cryopreservation of protocorm-like bodies ofDendrobium candidumby the air-drying method

Protocorm-like bodies (PLBs) of Dendrobium candidum were successfully cryopreserved by the air-drying method. The optimal water content before freezing seemed to be at the range of 0.1 g H₂O/g DW (11 % on fresh weight basis) to 0.5 g H₂O/g DW (33 % on fresh weight basis). Changes in soluble sugars, heat-stable proteins and dehydrins during desiccation of PLBs were analyzed. Extensive accumulation of soluble sugars was observed at water content of about 7.2 g H₂O/g DW (after 24 h desiccation), and the sugars content did not increase further during the following desiccation. The amount of heat-stable protein increased significantly when water content decreased to 1.0 g H₂O/g DW (after approximately 66 h desiccation). Results from immunological detection showed that two bands of the heat-stable proteins with respective molecular masses of 28.7 and 34.3 kDa were dehydrins which appeared when water content dropped to 1.0 g H₂O/g DW. Therefore, it seemed that accumulation of dehydrins happened later than that of soluble sugars. Interestingly, exogenous ABA treatment of PLBs before desiccation could also induce the accumulation of soluble sugars, heat-stable proteins and dehydrins. The possible roles of these substances in the acquisition of dehydration and freezing tolerance were discussed.     

Water relations of the freeze-tolerant New Zealand alpine cockroach Celatoblatta quinquemaculata (Dictyoptera: Blattidae)

Celatoblatta quinquemaculata is a freeze-tolerant alpine cockroach found on the Rock and Pillar Range, Central Otago, New Zealand. This study investigated seasonal changes in water content, as well as desiccation tolerance, and the relationship between desiccation and cold tolerance. Whole body water contents from field-fresh cockroaches collected over a 20 month period ranged from 69.9+/-1.0% fresh weight (FW) in February 1998 to 60.3+/-1.1% FW in July 1998. Water contents were significantly lower in winter than summer, and were positively correlated to microhabitat temperatures over the week preceding collection. Cockroaches survived the loss of up to 82% (mean: 56.7%+/-10.2) of their initial body water content, and the amount of water loss sustained was not dependent on the rate of water loss. Cockroaches did not suffer further mortality due to desiccation after removal to 99% relative humidity, but only regained lost water if given access to liquid water. Experimental dehydration did not enhance freeze-tolerance, but did slightly lower the supercooling point. It is concluded that reduction of body water content in winter may be a consequence of cold hardening responses, but desiccation does not constitute the cold hardening mechanism itself.     

Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L.

Summary Cryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.Abbreviations ANOVA analysis of variance - BA N6-benzyladenine - DMSO dimethyl sulfoxide - IBA indole-3-butyric acid - LN liquid nitrogen - MS Murashige and Skoog mineral solution - fwt fresh weight - LSD Least Significant Difference     

Increased Growth of the Microalga Chlorella vulgaris when Coimmobilized and Cocultured in Alginate Beads with the Plant-Growth-Promoting Bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.