The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifoliata (L.)

The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 g water per g dry mass is believed to increase cytoplasmic viscosity. Cooling to -196 degree C was done at rates averaging between 0.17 and 1300 degree C per second, and warming at 600 or 1.35 degree C per second. Survival was assessed after 4 weeks in vitro. Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70 percent normal development ranged between 0.17 and about 1300 degree C per second for axes at a water content of 0.26 g water per g dry mass narrowing with increasing hydration to an apparent optimum at about 686 degree C per second in axes at 0.8 g water per g dry mass At 1.7 g water per g dry mass, axes cooled at 0.17 degree C per second yielded nearly 40 percent normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.     

Cryopreservation of tea (Camellia sinensis L.) seeds and embryonic axes

This study investigated the tolerance to desiccation and freezing of tea seeds, embryonic axes (EAs) and cotyledonary embryonic axes (CEAs, consisting of EAs with portions of cotyledons still attached). No seeds germinated after desiccation and cryopreservation. EAs extracted from seeds desiccated to 18.9% moisture content (fresh weight basis) and cryopreserved showed 20.7% survival but plantlet production from these EAs was impossible. When EAs and CEAs were extracted from seeds before being submitted to desiccation and freezing, survival of control and frozen samples was equivalent with both types of materials. However, plantlet production was significantly higher from control and cryopreserved CEAs than EAs. The maturity stage of the seeds from which CEAs were extracted had an important effect on their survival and plant production percentages, mature seeds providing better results than early mature and late mature seeds. The highest percentages of plantlet production from cryopreserved CEAs, which ranged between 75.1 and 80.4%, were achieved for EA moisture contents between 21.5 and 15.0%.     

Cryopreservation of Quercus suber and Quercus ilex embryonic axes: in vitro culture,desiccation and cooling factors

This study examines different factors included in the cryopreservation protocols for Quercus ilex and Q. suber embryonic axes. In vitro incubation temperature played an important role in the appropriate development of Q. ilex axes, as 15 degrees C was superior to 25 degrees C. Q. suber axes proved to be more sensitive to desiccation and cooling. Poor survival (35%) was observed when axes were included into cryovials and then in liquid nitrogen, and none when immersed in sub-cooled liquid nitrogen (-210 degrees C). Q. ilex axes showed poorly organised development in vitro (c. 50% of non-cooled axes showed shoot development). However, c. 80% survival was observed after cryopreservation (either in liquid nitrogen or sub-cooled liquid nitrogen at 0.34 g water / g dry weight), of which c. 15% showed shoot development.     

Desiccation and freezing tolerance of embryonic axes from Citrus sinensis [L.] osb. pretreated with sucrose

Embryonic axes of Citrus sinensis L. were successfully cryopreserved. While fully hydrated unfrozen axes germinated 100%, survival decreased as axes water content dropped, and total loss of viability was observed when the water content dropped to 0.04 and 0.10 mg H2O/mg dry mass, for axes without and with sucrose preculture, respectively. Fully hydrated axes did not survive exposure to liquid nitrogen. Highest seedling recovery (93-100%) for untreated axes was observed at 0.26 to 0.15 mg H2O/mg dry mass. Differential scanning calorimetry revealed the presence of broad melting peaks in fully hydrated embryonic axes. The size of the melting peak diminished as water was removed by desiccation. Minimum melting of water was observed at the point axes survived cryopreservation. Occurrence of a glass transition upon warming was not a condition for axes to survive liquid nitrogen exposure. In untreated axes, glucose, increased with desiccation to 0.2 mg H2O/mg dry mass, and decreased as the axes were desiccated to lower water contents. Fructose and sucrose levels did not increase when untreated samples were desiccated for the same periods of time. Raffinose and stachyose levels decreased as untreated and precultured embryonic axes were desiccated. In sucrose precultured axes, sucrose and fructose levels increased when they were dehydrated, reaching maximum levels at 0.2 mg H2O/mg dry mass. Tissue glucose did not change significantly with desiccation. Raffinose and stachyose levels dropped as precultured embryonic axes were dried.     

15N allocation into wheat grains (Triticum aestivum L.) influenced by sowing rate and water supply at flowering under a Mediterranean climate

This study examined the effects of a reduced wheat sowing rate (250 vs. 500 grains m^–2) on grain yield, uptake of ¹⁵N into grains, and the incorporation into gluten and non-gluten proteins of wheat under field conditions in the Aegean region. A single ¹⁵N application was applied at stem elongation, at flowering, or at both developmental stages. Each ¹⁵N treatment included either additional water supply, or no additional water supply at flowering. Sowing rate (either 250 or 500 grains m^–2) had no impact on grain yield. Grain yield increased with additional water supply, but at the expense of protein quality, because of a decrease in the protein content of gluten. The ¹⁵N content of the gluten and non-gluten proteins at grain maturity was not different among cultivars. ¹⁵N applied at both stem elongation and flowering was found in comparable amounts in grains and protein fractions, irrespective of sowing rate.     

Kinetics of ultrasonic extraction of extractive substances from garden (Salvia officinalis L.) and glutinous (Salvia glutinosa L.) sage

The kinetics of ultrasonic extraction of extractive substances (ES) from dry herbs of garden (Salvia officinalis L.) and glutinous (Salvia glutinosa L.) sage using petroleum ether, 70% ethanol or water at 40 degrees C, as well as the composition of dry extracts, were studied. The mechanism of ultrasonic extraction is confirmed to occur in two steps: first, dissolution of the ES near the particle surface (washing) and, second, diffusion from the solid particles to the bulk of the liquid extract (slow extraction). The process is described mathematically using three concepts of the unsteady diffusion through plant material, the film theory and the empirical equation of Ponomaryov. The yield of ES increases with increasing solvent polarity, and nearly the maximum concentration of ES in liquid extracts is achieved for about 20 min. The composition of extracts depends on both the extraction conditions applied and the plant material.     

Investigation of the influence of cell density of human fibroblasts cryopreserved inside collagen sponges at various cooling rates

The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10(5) to 10(7) cells/cm(3). Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50 degrees C/min in a solution of Dulbecco's Modified Eagle Medium, 20% fetal bovine serum and 10% dimethylsulfoxide, kept frozen below -185 degrees C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.     

The peculiarities of water crystallization and ice melting processes in the roots of one-year plants (Plantago major L.)

Results are presented of a water phase transition study in plantain (Plantago major L.) roots, which were used as a model system to research the peculiarities of water crystallization and ice melting processes in complex heterogeneous biological systems. It was confirmed that water in such systems is crystallized in two clearly distinguished temperature ranges: -10 to -25 degree capital ES, Cyrillic and -25 to -45 degree capital ES, Cyrillic. These water fractions are conditionally attributed to extracellular (-10 to -25 degree capital ES, Cyrillic) and intracellular (-25 to -45 degree capital ES, Cyrillic) solutions. A possible explanation is given for such significant supercooling of the intracellular solution. The values of osmotic pressures of extra- and intracellular solutions were determined according to ice melting curves. It is noted that the intracellular solution, which crystallized at lower temperatures, had a lower osmotic pressure.     

Electrohydrodynamic (EHD) drying of rapeseed (Brassica napus L.)

The drying rate and germination parameters of rapeseeds (PF variety) treated by a high-voltage electrostatic field (HVEF) were investigated. The experiment was laid out as a factorial arrangement based on a completely randomized design with three replications. The results showed that the electrostatic field had a significant effect (P < 0.01) on decreasing rapeseed moisture content. Average drying rate for 8, 9, and 10 kV electrostatic fields over a time of 270 min increased by 1.78, 2.11, and 2.47 times, respectively, compared with that of the control. Drying rate increased with increasing voltage. Moreover, the results obtained from the germination experiment showed that the EHD had significant effects on the stemlet and radicle lengths of the rapeseed sprouts compared to that of the control. No significant effects were observed on the seed germination percentage and speed.     

Cryopreservation of mango (Mangifera indica L.) embryogenic cultures

Three techniques were compared for cryopreserving embryogenic masses (EMs) sampled from mango (Mangifera indica L.) cv. Zihua embryogenic cultures: (i) encapsulation/dehydration; (ii) pregrowth/dehydration; and (iii) vitrification. In all experiments, EMs were sampled from embryogenic cultures during their exponential growth phase and pretreated for 24 h on solid medium containing 0.5 M sucrose before freezing. No recovery was achieved after cryopreservation using the encapsulation/dehydration technique, whatever the moisture content (fresh weight basis) of EMs, which ranged from 78.3% without dehydration to 40.8% after 6 h dehydration. With the pregrowth plus dehydration technique, limited recovery (8.3%) was achieved after desiccation of EMs for 1 h, to 58.5% MC. Using the vitrification technique, recovery ranged from 94.3% after treatment of EMs with the PVS3 vitrification solution for 20 min (EM moisture content of 34.7%) to 10.9% after a 120 min treatment with the vitrification solution (EM moisture content of 26.0%).     

Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.     

紫外-可见分光光度法测定玉米不同部位籽粒早期发育的生理指标

为测定玉米不同部位籽粒早期发育的差异及氮素对籽粒发育的影响,实验用紫外-可见分光光度法对不同施氮水平下(0,120,180,240 kg·hm-2)玉米顶部和中下部籽粒在授粉后5～20 d的生理指标进行了检测.结果表明,施氮可明显促进籽粒可溶性总糖、蔗糖、淀粉含量的增加,促进蔗糖分解转化及淀粉合成关键酶活性的增强.当施氮量为180 kg·hm-2时,顶部籽粒的体积、干重及可溶性总糖、蔗糖、淀粉含量在授粉后20 d明显高于其他处理,酸性蔗糖转化酶AI、中性蔗糖转化酶NI、蔗糖合成酶SS、腺苷二磷酸葡萄糖焦磷酸化酶ADPGase及淀粉合成酶活性在授粉后5～20 d处于较高水平,使顶部籽粒的蔗糖利用能力及淀粉合成能力得到一定程度的改善,从而促进顶部籽粒发育,减少败育,提高产量.     

Ultrasonic extraction of oil from tobacco (Nicotiana tabacum L.) seeds

The ultrasonic extraction (UE) of oil from the seeds of a semi-oriental tobacco (Nicotiana tabacum L.) plant strain by using n-hexane and petroleum ether was studied at different temperatures and seeds-to-solvent ratios. The oil yield depended on the seed comminution, the extraction temperature, the seeds-to-solvent ratio and the type of solvent. The oil yield was much higher if the seeds were ground before extraction. The oil yield increased with increasing the extraction temperature and with decreasing the seeds-to-solvent ratio. n-Hexane was somewhat more efficient in the oil extraction than petroleum ether. In recovering the tobacco seed oil (TSO), the UE was less efficient than the Soxhlet extraction. The advantage of the UE was a relatively high oil yield at 25 degrees C in a shorter time. The kinetics of UE of TSO was described using the model of unsteady diffusion through plant material.     

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil

The effectiveness of ultrasonic-assisted extraction (UAE) of pomegranate seed oil (PSO) was evaluated using a variety of solvents. Petroleum ether was the most effective for oil extraction, followed by n-hexane, ethyl acetate, diethyl ether, acetone, and isopropanol. Several variables, such as ultrasonic power, extraction temperature, extraction time, and the ratio of solvent volume and seed weight (S/S ratio) were studied for optimization using response surface methodology (RSM). The highest oil yield, 25.11% (w/w), was obtained using petroleum ether under optimal conditions for ultrasonic power, extraction temperature, extraction time, and S/S ratio at 140 W, 40 °C, 36 min, and 10 ml/g, respectively. The PSO yield extracted by UAE was significantly higher than by using Soxhlet extraction (SE; 20.50%) and supercritical fluid extraction (SFE; 15.72%). The fatty acid compositions were significantly different among the PSO extracted by Soxhlet extraction, SFE, and UAE, with punicic acid (>65%) being the most dominant using UAE.     

Triboelectrification of houseflies (Musca domestica L.) walking on synthetic dielectric surfaces

Houseflies (Musca domestica L.) have been found to accumulate significant electrostatic charges when walking on uncharged dielectric surfaces. The number of steps taken was found to determine the amount of charge transferred whereas time, on its own, did not play a significant role. After walking only a short distance, typically 30 cm, flies reached saturation charge. The level of this varied according to the position of the surface in the triboelectric series relative to the fly. The rate of charging (pC/footstep) was directly proportional to the difference between the fly's charge and its saturation charge, hence the initial rate of charging for an uncharged fly was directly proportional to the saturation charge. A model has been fitted to the relationship between distance travelled (and hence the number of steps taken) with charge. The reciprocal charge left on the surface has been visualised using photocopier toner.     

Trace element composition of common sea buckthorn (Hippophae rhamnoides L.) tissues

Synchrotron radiation X-ray fluorescence analysis is used to determine for the first time the absolute content and biological accumulation coefficients for a set of 21 chemical elements (K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Zr, Nb, Y, Mo, and Pb) in the fruits, leaves, bark, roots, and root nodules of the sea buckthorn (Hippophae rhamnoides L. ssp. mongolica Rousi). It is found that all sea buckthorn tissues accumulate Ti, Nb, Cr, and Zr more intensively than the global average for terrestrial phytomass. On the other hand, none of the examined parts of the sea buckthorn plants accumulated the toxic elements Pb and As.     

Cryopreservation of garlic (allium sativum L.) using plant vitrification solution 2

Most cryopreservation procedures are optimized using a small number of germplasm accessions. We classified the garlic (Allium sativum L.) accessions that were selected for our studies based on genotype as identified using amplified fragment length polymorphism markers. Although recovery was variable, shoots regenerated from a broad range of the accessions after cryo-exposure. Garlic shoot tips were excised from cloves, surface sterilized, and placed on media at 5 degree C for 2 days prior to cryopreservation. Shoot tips were then treated with sucrose-glycerol for 20 minutes, plant vitrification solution 2 (PVS2; 15 percent w/v ethylene glycol, 15 percent w/v DMSO, 30 percent w/v glycerol, 13.7 percent w/v sucrose) at 0 degree C, and then plunged on foils into liquid nitrogen slush. Explants were recovered in 1.2 M sucrose for 20 minutes and then plated onto Gamborgs B5 medium containing alpha-naphthaleneacetic acid (NAA) and 6-(gammagamma-dimethylallylamino purine) (2-iP). Our results demonstrate that genotypically diverse accessions of garlic can be successfully cryopreserved.     

Comparison of classical and ultrasound-assisted isolation procedures of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.)

A comparative study of classical and ultrasound-assisted extraction and purification of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.), has been conducted. The isolated cellulose samples were studied by diffuse reflectance infrared Fourier transform spectroscopy and 13C nuclear magnetic resonance (13C-NMR) spectroscopy and the crystallinity was also determined. The use of ultrasound decreased the total time of treatment, in addition the purity of the obtained cellulose was very high.