Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration

Laccaria fraterna, a basidiomycetous, filamentous, non-sporulating (in vitro) fungus, was subjected to freeze-drying and tested for viability after rehydration. The optimization of the freeze-drying protocol included selection of the candidate fungus; optimizing physiological growth conditions and age; standardizing the protectant type and concentration; optimizing the pre-freezing method, freeze-drying run and extent of drying; choice of the rehydrant and the extent of rehydration. The culture retained its viability after lyophilization and when subjected to quality assurance tests gave consistent results similar to the non-lyophilized culture, indicating the stability of the product and the applicability of the developed process to freeze-dry vegetative mycelium of filamentous non-sporulating fungi.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cryoprotectant equilibration in tissues

The first step in the cryopreservation of cells or tissues is often the movement of a permeating cryoprotectant into the cells or tissues from the solution into which they have been placed. The cryoprotectant enters the cells or tissues by thermodynamic equilibration with the surroundings. In the reverse case, thermodynamic equilibration also drives the removal of permeating cryoprotectants by a dilution solution at the end of the preservation process when the cells or tissues are being readied for use. There have been reports of tissues having equilibrium cryoprotectant concentrations lower than that of the surrounding carrier solution. For various tissues, the equilibrium concentration of cryoprotectant inside the tissue is either equal to, or lower than the cryoprotectant concentration of the surrounding solution. A simple thermodynamic treatment of the solution-tissue equilibrium shows that an equilibrium concentration difference can exist between a tissue and the surrounding solution if a pressure difference can be maintained.     

寄主植物对甜菜夜蛾三龄幼虫抗寒力的影响

寄主植物是影响昆虫抗寒性的主要因子之一。研究了不同温度下甜菜夜蛾Spodoptera exigua(Hübner)3龄幼虫取食小白菜、甘蓝、葱和菠菜后,对过冷却能力和体内冷冻保护剂的影响。结果表明,寄主植物对甜菜夜蛾3龄幼虫的过冷却能力有显著性影响,其中以取食甘蓝的幼虫过冷却点最低。温度和寄主植物对其过冷却点、结冰点和虫体含水量有明显的交互作用。寄主植物对其体内的海藻糖含量有显著性影响,而对甘油和糖原含量没有显著性影响。温度和寄主植物仅对海藻糖含量有显著的交互作用。随着温度的升高,取食不同寄主的幼虫体内海藻糖和糖原含量的变化趋势完全相反,认为海藻糖是由糖原转化而来。研究结果提示冬季合适的寄主植物有利于甜菜夜蛾低龄幼虫越冬。     

飞蝗不同地理种群抗寒性研究

新疆和硕、哈密和天津北大港飞蝗种群的过冷却点随发育阶段而升高,在胚胎发育的各期都具有较低的过冷却点。飞蝗卵的过冷却点在这3个地理种群间无明显差异。雄性蝻和成虫双雌性的过冷却能力强,和硕飞蝗的成虫过冷点比北大港的飞蝗成虫要低。低温胁迫可诱导蝗卵以糖元为朱料合成抗冻保护剂,和硕飞蝗种群的卵主要合成甘油和山梨醇,北大港飞蝗种群的卵主要合成海藻糖,而哈密飞蝗种群的卵却可以合成甘油、山梨醇和海藻糖等抗冻保护剂。糖元磷酸化酶的总量随发育阶段而下降,但热休克和冷休克并不改变其总量,冷休克在胚胎发育的各阶段可以使其活性升高10%-40%,北大港飞蝗种群和哈密飞蝗种群的蝗卵经热休克2h处理后,糖元磷酸化酶的变化与在冷休克下的情况相似,而和硕飞蝗处群胚胎发育的Ⅱ期,热休克诱导糖元磷酸化酶活性的升高程度较低。     

Survival of Heleomyza borealis (Diptera, Heleomyzidae) larvae down to − 60°C

Heleomyza borealis Boh. (Diptera, Heleomyzidae) overwinters as larvae in Arctic habitats, where they may experience winter temperatures below ? 15°C. The larvae freeze at c.? 7°C but in acclimation experiments 80% survived when exposed to ? 60°C. Of the larvae exposed to between ? 4 and ? 15°C, only 3% pupated. However, when cooled to ? 20°C this increased to 44%, with 4% emerging as adults. Larvae maintained at 5°C contained low levels of glycerol, sorbitol and trehalose, which did not increase with acclimation to low temperatures. However, levels of fructose increased from 6.1 μg mg^?1 fw in control animals to 17 μg mg^?1 fw when exposed to ? 2°C for 1 week. Larval body water (2.2 ± 0.1 g/g dw, mean ± SD, n = 100) and lipid content (0.22 ± 0.002 g/g fw, mean ± SE) showed no significant change during acclimation to low temperatures. Larvae maintained at a constant 5°C survived for over 18 months with little loss of body mass (from 7.5 ± 1.2 to 7.0 ± 1.2 mg fw, mean ± SD, n = 20), but none pupated. Heleomyza borealis larvae appear to feed and grow until they reach a body mass of about 7.5 mg and then become dormant. They remain in this state until they experience a low temperature stimulus (< ? 15°C) followed by a warm period (≈ 5°C). This ensures that the larvae pupate and adults emerge in early summer, allowing the maximum growing period before the following winter. Heleomyza borealis are adapted to survive long winters in a dormant larval state. They have a low metabolic rate, can conserve body water even at subzero temperatures but do not synthesize large quantities of cryoprotectants.     

Long-Term Storage of Infective Microsporidian Spores in Liquid Nitrogen

Thirty-one species of microsporidia, isolated from insects and stored in liquid nitrogen for up to 25 yr, were infectious when removed from liquid nitrogen. The natural hosts of all of these microsporidia were terrestrial insects, representing six different insect orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera. All microsporidia from terrestrial insects that were tested survived storage in liquid nitrogen, while Nosema algerae , a microsporidium from aquatic mosquito hosts did not survive freezing in liquid nitrogen. A Nosema species from the alfalfa weevil, Hypera postica , lost some infectivity in a water storage medium after 25 yr in liquid nitrogen. Liquid nitrogen storage of microsporidian spores in 50% and 100% glycerol media reduced loss of infectivity and is recommended for extended storage of microsporidia from terrestrial insect hosts.     

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.     

Metabolism and cold tolerance of Chinese white pine beetle Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) during the overwintering period

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Equilibrium,quasi-equilibrium,and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.