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Liquid nitrogen storage of yeast cultures. I. Survival, and literature review of the perservation of fungi at ultralow temperatures.

All 20 yeast strains of 17 species tested survived 75 days (the length of the experimental period) in liquid nitrogen at -196 C. The components of the more protective of the two freezing media used were (w/v) malt extract 2.5%, yeast extract 0.25%, peptone 0.5%, calf serum 15% (v/v) and dimethyl sulfoxide 10% (v/v). Viability of the cells in this medium after rapid uncontrolled freezing and thawing in selaed plastic ampoules ranged from 2% to 98% (average 67%) compared with the viability of the cultures before freezing. In only 4 strains was survival lower than 50%. (90 references).

Culture Media↗

Compact liquid nitrogen storage system yielding high recoveries of gram-negative anaerobes.

A simple and compact system suitable for the preservation of fragile gram negative anaerobes and other bacteria in liquid N2 has been developed. Polypropylene straws used as specimen containers can be used easily within glove bags of anaerobic chambers, and their small size greatly increases the number of cultures which can be stored. Ancillary equipment and methods developed are described. The overall system was tested, using Streptococcus mutans, Fusobacterium nucleatum, and Selenomonas sputigena. Various basal suspending fluids and cryoprotective supplements were studied. With fast rates of freezing and thawing, survival recoveries of the test microorganisms ranged from 80 to 100 percent of the input colony-forming units in a complex medium broth base without cryoprotective agent addition, and they consistently were 100 percent when 0.4 mM polyvinylpyrrolidine was used. Overall, cryoprotection by polyvinyl pyrrolidine was superior to that from glycerol or dimethyl sulfoxide, the latter yielding recoveries similar to or less than those obtained with no cryoprotectant additive. All microorganisms were recoverable after storage for 1 year.

Dimethyl Sulfoxide↗

Soil carbon and nitrogen storage in response to fire in a temperate mixed-grass savanna.

Vegetation fires may alter the quantity and quality of organic matter inputs to soil, rates of organic matter decay, and environmental factors that influence those processes. However, few studies have evaluated the impacts of this land management technique on soil organic carbon (SOC) and total N in grasslands and savannas. We evaluated the impact of repeated fires and their season of occurrence on SOC and total N storage in a temperate mixed-grass-mesquite savanna where fire is used to control woody plant encroachment. Four fire treatments varying in season of occurrence were examined: summer only (SF), winter only (WF), alternate summer and winter fires (SWF), and unburned controls. In each treatment, soils were sampled to 1 m under three vegetation types: C3 grasses, C4 grasses, and mesquite trees. The SOC storage at 0 to 20 cm was significantly greater in SF (2693 g C m(-2)) and SWF (2708 g C m(-2)) compared to WF (2446 g C m(-2)) and controls (2445 g C m(-2)). The SWF treatment also increased soil total N (271 g N m(-2)) relative to all other treatments (228-244 g N m(-2)) at 0 to 20 cm. Fire had no effect on SOC or total N at depths of > 20 cm. Vegetation type had no significant influence on SOC or total N stocks. The delta13C value of SOC was not affected by fire, but increased from -21 per thousand at 0 to 10 cm to -15 per thousand at depths of > 20 cm indicating that all treatments were once dominated by C4 grasses before woody plant encroachment during the past century. These results have implications for scientists, land managers, and policymakers who are now evaluating the potential for land uses to alter ecosystem C storage and influence atmospheric CO2 concentrations and global climate.

Carbon↗

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: identification, characterization and immunolocalization of a putative taproot storage glycoprotein.

In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.

Abscisic Acid↗

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: effects of methyl jasmonate on nitrate uptake, senescence, growth, and VSP accumulation.

The role of methyl jasmonate (MeJa) in promoting senescence has been described previously in many species, but it has been questioned in monocarpic species whether induced senescence is a result of a potential death hormone like MeJa, or a consequence of an increased metabolic drain resulting from the growth of reproductive tissue. In oilseed rape (Brassica napus L.), a polypeptide of 23 kDa has been recently identified as a putative vegetative storage protein (VSP). This polypeptide could be used as a storage buffer between N losses from senescing leaves putatively promoted by methyl jasmonate that might be produced by flowers, and grain filling which occurs later on, while N uptake is strongly reduced. In order to describe causal relationships during Brassica napus L. plant responses to MeJa treatment, a kinetic experiment was performed to determine the order and the amplitude with which general processes such as growth, photosynthesis, chlorophyll content, N uptake, and N storage under the form of the 23 kDa VSP are affected. One of the most immediate consequences of MeJa treatment was the strong reduction of nitrate uptake within 6 h, relative to control plants. However, this was not a specific effect as K(+) uptake was similarly affected. Photosynthesis was reduced later (after 24 h), while chlorophyll content as well as leaf growth also decreased in a similar way. Moreover, this was concomitant with a remobilization of endogenous unlabelled N from senescing leaves to roots. Accumulation of the 23 kDa VSP was induced in the taproot after 24 h of MeJa treatment and was increased 10-fold within 8 d. On the other hand, the reversible effect of a MeJa pretreatment was tested in the long term (i.e. along the growth cycle) using plants previously grown in field conditions induced for flowering. Results show that a MeJa pulse induced a reversible effect on N uptake inhibition. In parallel, protein immunologically related to the 23 kDa VSP was detected in stems with a similar molecular weight (23 kDa), and in flowers and leaves with a molecular weight of 24 kDa. This accumulation was concomitant with the remobilization of both subunits of Rubisco. During stem and pod development, this protein induced by MeJa is fully hydrolysed. The external and intermittent supply of MeJa mimic some of the plant physiological processes previously reported under natural conditions. This suggests that in oilseed rape, methyl jasmonate could be considered as a possible monocarpic senescence factor while accumulation/mobilization of the 23 kDa VSP in taproot could be a marker for the cessation of N uptake and the initiation of a massive leaf senescence.

Acetates↗