Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 M abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 M ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.Abbreviations ABA abscisic acid - PEG polyethylene glycol - TAG triacylglycerol - TL total lipid - TEM transmission electron microscopy Plant Research Centre contribution No. 1383We are grateful to Dawn Moore and Ken Stanley for technical assistance, and thank Pat Rennie for the electron microscopy. We acknowledge financial support through an NSERC/Forestry Canada/Weyerhaeuser Canada Ltd (Prince Albert, Sask.) research partnership programme.     

Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells

Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of ⁸⁶Rb was generally decreased by increasing concentrations of Ca²⁺, but after longer periods of incubation, the inhibiting effect of high Ca²⁺ levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at −1°C, while in the presence of 10 millimolar Ca²⁺ viability declined only gradually over a 5-week period. Ice encasement markedly reduced ⁸⁶Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from −1 to −2 and −3°C, but the presence of Ca²⁺ in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from −10 to −30°C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at −1°C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane.     

Metabolic and Ultrastructural Changes in Winter Wheat during Ice Encasement Under Field Conditions

The effect of ice encasement on the physiological, metabolic, and ultrastructural properties of winter wheat (Triticum aestivum L.) grown under field conditions was examined by artificially encasing winter wheat in ice during early winter. Cold hardiness and survival of ice-encased seedlings declined less rapidly in Kharkov, a cold-hardy cultivar than in Fredrick, a less hardy cultivar. Ethanol did not accumulate in non-iced seedlings, but increased rapidly upon application of an ice sheet. Lactic acid accumulated in both cultivars during late autumn, prior to ice encasement, and elevated levels of lactic acid were maintained throughout the winter in seedlings from both iced and non-iced plots. The rate of O₂ consumption of shoot tissue of seedlings from non-iced plots remained relatively constant throughout the winter, but declined rapidly in seedlings from ice encased plots. Major ultrastructural changes did not occur in shoot apex cells of non-iced winter wheat seedlings during cold hardening under field conditions. However, the imposition of an ice cover in early January resulted in a proliferation of the endoplasmic reticulum membrane system of the cells, frequently resulting in the formation of concentric whorls of membranes, often enclosing cytoplasmic organelles. Electrondense areas within the cytoplasm which appeared to be associated with the expanded endoplasmic reticulum were also frequently observed.     

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse,rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970)208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F^?, malonate, and arsenite), only F^? (100 mm final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.     

Metabolic and ultrastructural changes associated with flooding at low temperature in winter wheat and barley

Cold-hardened winter wheat (Triticum aestivum L. cv. Fredrick) and winter barley (Hordeum vulgare L. cv. Dover) were exposed to total flooding at 2 C. Dover seedlings were damaged more quickly than Fredrick, and after 3 weeks of flooding the survival of Dover was reduced to 10% and Fredrick to about 50%. Tissue moisture was slightly greater in Dover than Fredrick throughout the 4-week flooding period. Carbon dioxide and ethanol accumulated throughout the 4-week flooding period in both cultivars. Lactic acid increased rapidly during the 1st week of flooding, and remained relatively constant during the remainder of the flooding period. Oxygen consumption of seedling shoot tissue after exposure to flooding declined abruptly after only 1 day of flooding, but recovered somewhat during the subsequent 2 weeks. The effect of flooding was more pronounced on the ultrastructure of Dover than Fredrick. Although proliferation of endoplasmic reticulum was observed in the early stages of flooding in both cultivars, the occurrence of distinct parallel arrays and concentric whorls of membranes was prevalent in the flooded barley. Severe ultrastructural damage to a large proportion of apical cells in both cultivars was observed after 2 to 3 weeks of flooding.     

Finescale ecological niche modeling provides evidence that lactating gray seals (Halichoerus grypus) prefer access to fresh water in order to drink

Many phocids are capital breeders, relying on stored reserves to sustain energetic requirements while on land. Their large body size, high energy expenditure during lactation, and the insulative effects of the blubber layer can lead to thermal stress from overheating, especially in warm and temperate climates. Thermal stress can influence fine‐scale site choice on breeding colonies, and behavioral thermoregulation has been proposed as an explanation for the clear preferences shown by breeding female gray seals for proximity to pools of water. However, anecdotal observations suggest that pools of water may also be preferred for drinking, though water intake is difficult to verify without real‐time physiological monitoring. Here, an alternative approach demonstrates that gray seals also require access to water for drinking. Using Ecological Niche Factor Analysis to examine fine‐scale physical determinants of pupping site choice at North Rona, Scotland, we found that lactating mothers showed preference for lower salinity pools. This is most pronounced early in the season, when ambient temperatures and presumably thermal stress are greatest. Given that the cooling effect of fresh and salt water should be equivalent, the most parsimonious explanation for this preference for fresh water pools is that lactating females use these pools for drinking.