Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.     

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.     

Cryopreservation of embryogenic tissues of Pinus nigra Arn. by a slow freezing method

Six different embryogenic cell lines of Pinus nigra Arn. have been cryopreserved in liquid nitrogen using cryoprotection with sucrose (18%) and DMSO (7.5%). Post-thaw growth and tissue proliferation have been observed in five cell lines. The survival levels after storage in liquid nitrogen reached values between 62.5 and 100%. Growth of recovered embryogenic cells as well as somatic embryos is similar to the non-frozen tissues maintained in long-term culture. Somatic embryo maturation and plantlet regeneration occurred in all selected cell lines.     

Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cell suspensions

The freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cells, as determined by electrolyte leakage, was increased by the incubation of samples in medium containing 0.8 M sucrose. To elucidate the mechanism involved, we investigated the changes in soluble carbohydrates, cell ultrastructure and proteins accompanying the increase in freezing tolerance following incubation in sugar-rich medium. During sugar incubation, the intracellular sucrose content increased from 67 mol g-1FW to 429 mol g-1FW; it was also metabolized into fructose and glucose, as determined by high-performance liquid chromatography. Microscopy revealed that sugar incubation induced plasmolysis of embryogenic cells and drastic changes in cell ultrastructure with the appearance of rough endoplasmic reticulum (rER). Furthermore, immunoblotting analysis with anti-dehydrin antiserum revealed that a dehydrin-like protein appeared only when maximal freezing tolerance was induced by sugar incubation. These results suggest that freezing tolerance of asparagus embryogenic cells is increased by a complex mechanism involving notably changes in cell ultrastructure and accumulation of certain sugars and proteins during sugar incubation.     

Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.     

Cryopreservation of Pistacia spp. seeds by dehydration and one-step freezing

Cryopreservation protocols by dehydration and one-step freezing were developed for seeds from three Pistacia species, i.e., P. vera, P. terebinthus and P. lentiscus, which were characterised by different initial germination percentages (100%, 17% and 81%, respectively). In P. vera, a maximum of 90% germination was obtained following 8 hours drying in silica gel (corresponding to 11.7% moisture content on a FW basis) and direct immersion in LN. In P. terebinthus and P. lentiscus, shorter periods of dehydration (1 hour and 15 min, respectively) were sufficient to reduce their moisture content to about 20%, which resulted in peak seed germination percentages from cryostorage of 16% and 47%, respectively. Following cryopreservation, the seeds germinated better on semi-solid MS medium, than on cotton wool wetted with dH(2)O or liquid MS medium. Finally, in P. vera and P. lentiscus, high and significant correlation coefficients were obtained between the TTC viability test and seed germinability after recovery from LN, provided that seeds which were considered positive in the test showed completely or partially red embryonic axes coupled to completely red cotyledons.     

Cryopreservation, encapsulation and promotion of shoot production of embryonic axes of a recalcitrant species Ekebergia capensis, Sparrm

A study on zygotic axes of the recalcitrant seeds of Ekebergia capensis compared two cryopreservation methods, partial desiccation, and encapsulation-dehydration, and also investigated a method to promote shoot production. High (80 percent) survival (assessed as root production) was obtained after direct immersion into liquid nitrogen of axes rapidly dehydrated by flash drying for 20 min to a water content about 0.4 g water per g dry mass. In contrast, no survival at all was obtained of axes that were first encapsulated, then desiccated for three hours to the same water concentration as those fast-dried, and then placed in a cryovial and immersed in liquid nitrogen. Axes encapsulated after cryopreservation germinated both in vitro and in soil, and could be stored at room temperatures for several weeks while maintaining germinability, thus producing synseeds capable of distribution. However, shoot production after cryopreservation was seldom observed. The inclusion of the plant growth regulator, N6-benzyl adenine (BA) in the MS-based recovery medium promoted vigorous multiple shoot formation. Microscopical examination of embryos of E. capensis revealed that the cotyledonary insertions were contiguous with the shoot apex, leading to the conclusion that injury to, and ultimate necrosis of, the apical meristem following severing of these connections was a primary cause of the observed lack of, or poor, shoot development in excised axes (whether cryopreserved or not). The study demonstrated that it may be possible to resolve two of the problems facing attempts at cryopreservation of axes of recalcitrant seeds; lack of shoot production and difficulty of distribution of cryopreserved material for re-introduction.     

微波消解-火焰原子吸收光谱法测定秦艽和麻花秦艽中多种微量元素

密封微波消解已被运用于火焰原子吸收光谱法对中草药中微量元素含量的测定过程中。微波消解法具有方便省力,安全快捷,污染少,样品溶解完全等特点。选用硝酸和双氧水（5∶1）的混合消化液作为消解剂进行微波消解。运用火焰原子吸收光谱法测定了秦艽和麻花秦艽中的Fe,Mn,Ni,Cu,Zn,Ca,Mg,Cr等八种微量元素的含量。进行了微波消解条件的选择及消化结果精密度实验,该方法的加标回收率为88.1%～114.5%,相对标准偏差（RSD）<3.12%,具有较为良好的准确度和精密度。结果表明,秦艽和麻花秦艽均富含Ca,Mg,Fe,Mn,Zn等元素,而Ni和Cr含量均较低。通过对秦艽和麻花秦艽微量元素含量的比较,发现秦艽中的Mg, Fe, Mn, Ni含量较高,而麻花秦艽中的Zn, Cu, Ca, Cr含量较高。此结果为探讨微量元素与秦艽和麻花秦艽的药效关系提供了一定的科学数据。并且,通过本实验为秦艽和麻花秦艽的进一步研究和综合开发利用提供了新的科学依据。     

Cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus spp.) by vitrification

An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 microM BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25 degree C. Clumps that grew many buds were cold-hardened at 5 degrees C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5 degrees C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25 degree C. Then they were dehydrated in 1 ml PVS2 solution at 25 degree C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa 'NZ219' buds reached 87 percent. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30 to 90 percent and an average of 63 percent.     

Characterization of suspensions of particles in water by an ultrasonic resonant cell

This paper presents a resonant technique to accurately measure phase-velocity and attenuation of longitudinal acoustic waves in suspensions of solid particles in water. The technique is based on exciting thickness resonances of a layer of fluid and analyzing its spectrum. To this end, a resonant cell to contain the fluid is described and used. Two different type of water suspensions are studied: titanium dioxide and alumina particles; particle volume fraction is in the range 0–0.18. Simultaneous determination of particle size distribution in the suspension by an optical method are also carried out. Finally, the experimental results are compared with theoretical predictions obtained from three different approaches.     

Comparison of cell membrane water permeability in monolayers and suspensions

We previously measured the membrane water permeability of monolayers and suspensions of MIN6 mouse insulinoma cells at room temperature, and found that water transport was faster in monolayers. Here, we compare water transport kinetics in monolayers and suspensions over a range of temperatures for two different cell types, MIN6 cells and bovine pulmonary artery endothelial cells (BPAEC). At room temperature the results for BPAEC and MIN6 cells were similar, with approximately 2-fold faster water transport in monolayers than suspensions. The activation energy for water transport (Ea) was estimated from Arrhenius plots of the water permeability data. The values of Ea for monolayers and suspensions of MIN6 cells were not significantly different. However, the activation energy was significantly lower for BPAEC monolayers (Ea = 49 +/- 2 kJ per mol) than suspensions (Ea = 70 +/- 4 kJ per mol). Predictions of water transport during cryopreservation revealed substantial differences in supercooling between monolayers and suspensions.     

The DNA content of mouse two-cell embryos can be measured by microfluorimetric image analysis under conditions of cell viability

Video-enhanced fluorescence imaging was used to quantify the DNA content in live two-cell mouse embryos. DNA was stained with the vital fluorophore Hoechst 33342. Conditions of dye concentration and irradiation were such that two-cell embryos could be kept in the constant presence of the dye for about 24 h without a major effect on their furtherin vitro viability. Total nuclear fluorescence intensity of stained two-cell embryos was measured twice under these conditions, i.e., in G1 (1 h after cleavage) and in G2 (15–18 h after cleavage), by image analysis. After correcting for the fluctuations in excitation intensity and for the spatial nonhomogeneities of the optical system (lenses and sensor), the mean total nuclear fluorescence intensity was about twofold higher in G2 than in G1 (R=1.99 to 2.25), and this increase was abolished by the addition of aphidicolin, an inhibitor of replication. The fluorescence increase did not depend on the Hoechst concentration in the range of 10–40 ng/ml, i.e., in the range of embryo viability. The coefficient of variation of the total nuclear fluorescence intensity measured under these conditions was rather large (10 to 20%). Nevertheless, the mean value of fluorescence intensity in G1 of nuclei of a given pool represents an appropriate reference to measure the increase in fluorescence intensity between G1 and G2.     

Quantification of water compartmentation in cell suspensions by diffusion-weighted and T(2)-weighted MRI

When studying water diffusion in biological systems, any specific signal attenuation curve may be reproduced by a broad range of mathematical functions. Our goals were to quantify the diffusion and T₂ relaxation properties of water in a simple biological system and to study the changes that occur in osmotically stressed cells.     

透光脉动检测仪研究碱度对絮体形成、破碎及再生的影响

为了解碱度对絮体的形成、破碎及再生过程的影响,采用PDA2000型透光脉动检测仪测定不同碱度下投加硫酸铝时高岭土悬浮液的絮凝指数(FI指数),并以强度因子和再生因子评价絮体的强度和再生能力。结果表明,碱度的高低在很大程度上会影响絮体的形成、破碎及再生过程,所形成絮体的颗粒随碱度的增加而减小。混凝剂投量和碱度高低共同决定了絮体的抗破碎强度,碱度越高,混凝剂投量越大,絮体的强度越高。在电中和作用下形成的絮体在低碱度下经一次破碎后恢复程度接近100%,在网捕卷扫作用下形成的絮体,无论碱度高低,从第2次破碎起,FI指数均以大于10%的幅度逐次下降,絮体不可恢复程度显著增大。     

Device for aeration and mixing of cell and organelle suspensions during NMR experiments

A device for aeration and mixing of cell or organelle suspensions in a vertical bore NMR magnet is described. Multiple external sensors (e.g., ion-selective electrodes) may be immersed in the suspension within the bore of the magnet. The sensors are positioned to avoid noise due to contact with gas bubbles and proximity to the probe head. The required sample volume is minimised. The modular design of components permits the use of the device in magnets of various internal dimensions, or with probe heads of different sample tube diameter, by modification of the simpler components of the assembly.     

Flow, ordering, and jamming of sheared granular suspensions

We study the rheological properties of a granular suspension subject to constant shear stress by constant volume molecular dynamics simulations. We derive the system "flow diagram" in the volume fraction or stress plane (phi, F): at low phi the flow is disordered, with the viscosity obeying a Bagnold-like scaling only at small F and diverging as the jamming point is approached; if the shear stress is strong enough, at higher phi an ordered flow regime is found, the order-disorder transition being marked by a sharp drop of the viscosity. A broad jamming region is also observed where, in analogy with the glassy region of thermal systems, slow dynamics followed by kinetic arrest occurs when the ordering transition is prevented.     

基于FTIR技术和稀疏线性判别分析的秦艽种类鉴别

傅里叶变换红外光谱通常包含有大量的波长变量点,对其进行定性分析需要建立稳健的、可解释性的分类模型。稀疏线性判别分析(SLDA)是一种较为新颖和有效的机器学习算法,常用于高维度、小样本数据的变量筛选和判别分析,SLDA通过在线性判别分析中引入正则项,使分类器训练过程和变量选择过程同时完成,不同判别方向上载荷系数的稀疏性则增强了模型的可解释性。采集甘肃不同产地的秦艽样本94个,其中麻花秦艽(Gentiana straminea Maxim)30个,黄管秦艽(Gentiana officinalis)28个,大叶秦艽(Gentiana macrophylla Pall)36个,利用傅里叶变换红外光谱法获得所有样本的光谱图。取其中70个样本构成训练集,剩余24个为测试集。使用训练集建立SLDA模型,对2个判别方向上不为0的载荷系数个数进行网格化寻优,得到了最优的参数空间。利用建立的SLDA模型对测试样本进行预测,其分类准确率达到100%,实现了对三种秦艽的快速、准确鉴别。实验结果表明,与PLS-DA方法相比,SLDA模型在分类准确率、稀疏性及可解释性方面均具有一定优势,是一种新颖、有效的光谱定性分析方法。     

Collapse temperature of bacterial suspensions: the effect of cell type and concentration

The characterisation of the physical state of frozen and freeze dried biological products delivers powerful information for freeze-drying process optimisation. The influence of lactic acid bacterial cell size, shape and concentration on collapse temperature of concentrated bacterial suspensions was investigated. Lactobacillus bulgaricus (long rods), and Streptococcus thermophilus (small spherical cells) were used as cellular models for this study. Whatever the strain, when lactic acid bacterial cells were added to protective solutions, the collapse temperature increased, thus allowing the use of higher sublimation temperatures during primary drying than expected from the protective medium alone. Moreover, the higher the cell concentration, the greater the effect, linear relationships existing between the collapse temperatures and the total dried matter. Cells of both strains gave a kind of robustness to the freeze-dried product, but the increase observed in collapse temperature was considerably higher (3 - 5 degree C) for L. bulgaricus compared to S. thermophilus. This result was ascribed to the different size and shape of the strains.     

Human T-cell line (Jurkat cell) encapsulation by nano-organized polyelectrolytes and their response assessment in vitro and in vivo

Journal of Nanoparticle Research - Silver nanoparticles (AgNPs) are of interest due to their antimicrobial attributes, which are derived from their inherent redox instability and subsequent release...