Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.     

New determinants for tolerance of coffee (Coffea arabica L.) seeds to liquid nitrogen exposure

The present work establishes for the first time that tolerance of coffee seeds to liquid nitrogen (LN) exposure depends on the initial quality of the seedlot and on the rewarming regime employed. Seedlot quality was estimated by the parameters of a quantal response model of desiccation sensitivity developed previously. The percentage of seedlings recovered from cryopreserved seeds was very well correlated with the relative humidity (RH) at which 90 percent of the initial viability was retained, RH90, as estimated by the model. Whatever the cooling regime employed, rewarming the seeds slowly by exposing them to ambient air was highly detrimental. Slow rewarming-induced viability loss was not due to imbibitional damage since seeds pre-heated at 37 degree C after slow rewarming to 0 degree C exhibited a survival percentage lower than seeds thawed rapidly to 0 degree C before sowing. The optimal hydration status for coffee seed cryopreservation was also re-examined. Drying seeds in 81 percent RH provided survival percentages considerably higher than those obtained using the drying RH always employed until now, i.e. 78 percent. A new procedure for slowly precooling the seeds prior to immersion in LN was also established. It consisted of placing the vials containing the seeds in a dry ice-bath for 25 min. Using this procedure in combination with seed drying in 81 percent RH and rapid rewarming in a 37 degree C water-bath for 30 min ensured the highest survival percentages ever obtained with coffee seeds, i.e. 89 percent, a value which was not significantly different from the initial viability percentage.     

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.     

Relative angular precision in electron backscatter diffraction: A comparison between cross correlation and Hough transform based analysis

In the present study the relative angular resolution of an electron backscatter diffraction system based on Hough transform analysis has been determined with a silicon single crystal wafer. The resolution is found to be better than 0.1° and can be easily improved by repetition of measurements. A test measurement on a BaFe₂As₂ thin film, where disorientations of 0.1° and less are present, was performed using the cross correlation electron backscatter diffraction technique. The same measurement is evaluated with the Hough transform technique. Comparing both techniques give evidence of a relative resolution of better than 0.1°. However, in specimen areas with strain inhomogeneities a deviation along one rotation axis can be observed.     

Development and validation of a HPAE-PAD method for the quantification of CGP69669A, a sialyl Lewis(x) mimetic, in skin permeation studies

A simple, rapid, precise and specific isocratic HPAE‐PAD method for quantification of CGP69669A was developed and validated. CGP69669A is a glycomimetic of sialyl Lewis^x and an antagonist of E‐selectin with potential application in the treatment of inflammatory skin disease. Quantification was performed using a Dionex CarboPac^TM PA‐200 anion‐exchange column (3 × 250 mm) with 100 mm NaOH solution as mobile phase, a flow rate of 0.50 mL/min and an injection volume of 10 μL. A quadruple potential waveform was used to detect the carbohydrate (+0.1 V from 0.00 to 0.40 s, ?2.0 V from 0.41 to 0.42 s, +0.6 V at 0.43 s and ?0.1 V from 0.44 to 0.50 s with current integrated between 0.20 and 0.40 s for detection) and rafinose was employed as an internal standard. The optimized conditions enabled rapid elution of CGP69669A (at 3.0 min) without interference from solvent peaks or substances present in the skin. The method showed good intra‐ and inter‐day precision and accuracy and the response was linear from 1.0 to 25 µg/mL. This is the first validated direct method for the quantification of CGP69669A. It will now be employed in studies investigating the topical and transdermal delivery of CGP69669A in vitro and in vivo and it should also be of use for other applications of this molecule. Copyright © 2011 John Wiley & Sons, Ltd.     

Glycosynthases as tools for the production of glycan analogs of natural products

Oligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field.     

Quality control and analytical test method for Taxus baccata tincture preparation

The homeopathic tincture of Taxus baccata L. is monographed in the current German Homeopathic Pharmacopoeia (HAB 2009). However, the described identification test is a common comparative TLC procedure that might be updated. The purpose of the current work was the quali-quantitative analysis by HPLC/DAD/MS of Taxus tincture. In this study we characterized polyphenolic compounds, in particular four hydroxycinnamic derivatives (0.85 mg/mL) and four flavonoids (quercetin and kaempferol 3-O-rutinoside and xylosyl glucosides); the total polyphenol content was 1.265 mg/mL of tincture. Starting from the official German Homeopathic Pharmacopoeia method of preparation, the aim of this work was to optimize a rapid and reproducible method for the analysis of herbal drugs and tincture, directly prepared in store or the herbalist's shop, to ensure safety and efficacy of the preparation. The procedure has to ensure validation, robustness of the results, and provide a quick response about the composition of compounds in the herbal drug preparation.     

Polyaniline nanofiber–silica composite aerogels

Strong, electrically conducting aerogels were prepared by introducing polyaniline nanofibers to a silica sol just prior to gelation and drying through supercritical carbon dioxide processing. The addition of a few milligrams of polyaniline per cm³ increased the flexural strength of the cylindrical monoliths by 200%. Using preformed polymeric nanofibers avoided filling of microporosity often observed with polymer reinforcement of aerogels and allowed preparation of polyaniline–silica composite aerogels with surface areas over 900 m²/g. Despite the small amount of polyaniline nanofibers (1.3–16.5 wt.%), the composite aerogels were electrically conducting (8.0 × 10^? 8–1.83 × 10^? 5 S/cm) and it was possible to prepare chemiresistor sensors for detection of acidic (HCl) and basic (ammonia) gaseous molecules with response times similar to thin film sensors containing orders of magnitude more polyaniline.     

Dual-functional probes towards in vivo studies of brain connectivity and plasticity

A Gd(3+) based paramagnetic dextran conjugate has been developed, which enables the tracking of neuroanatomical connectivity in the brain by both MR and optical imaging. Cell studies and subsequent in vivo experiments in rodents demonstrate efficient internalisation and transport properties of the new tracer molecule.