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A novel method for the visualization and quantification of the state of dispersion of calcium carbonate particles in thin blown polymer films is described. Particle imaging was achieved by elemental mapping using energy dispersive spectroscopy. This generated outlines of particles and agglomerates located close to the film surface. ImageJ software facilitated the extraction of the corresponding Feret diameters. Finally, the Bootstrap technique was used to estimate confidence intervals for the kurtosis of the Feret particle size distribution. Kurtosis is a statistic that describes the shape of a distribution’s tails in relation to its overall shape. It therefore provides a measure that characterizes the degree of particle agglomeration. The proposed procedure was applied to analyze high-density polyethylene films prepared using different calcium carbonate masterbatches in which formulation parameters were varied.

The effects of filler content, filler particle size and surface coating, as well as the natures of the wax and metal stearate lubricants on the properties of calcium carbonate-filled linear low-density polyethylene compounds were investigated. The elongation at break decreased exponentially with filler loading irrespective of whether the filler was surface coated or not. The corresponding decline in the impact strength was more gradual but it dropped abruptly on reaching a filler loading of 70 wt%. The effect of shear rate on the apparent melt viscosity followed an empirically modified Carreau–Yasuda model which included the effect of filler content. It features the zero-shear viscosity and the flow consistency index as adjustable parameters. These characterize the viscosity trends at low- and high-shear rates, respectively. In the absence of additives, the melt viscosity at 60 wt% filler exceeded that of the neat polymer by a factor of three. Adding 3 wt% wax and 1.0 wt% zinc stearate returned the melt viscosity to levels just above that of the neat polymer. Calcium stearate and magnesium stearate were less effective at reducing the apparent melt viscosity but a blend of the latter with zinc stearate performed particularly well.

Malaria remains a public health concern with vector control still the vital component of disease prevention, control, and elimination strategies. Recent years has seen a “stalling” in the progress ...

This study aimed to develop cellulose-based polymer matrices as controlled release devices for plant-based insect repellents and attractants, with the aim of finding sustainable and environmentally friendly pest control methods for agricultural applications. Citronellol, terpineol and methyl salicylate were the selected active compounds for this study. Their compatibility with cellulose diacetate was predicted using Hansen Solubility Parameters, which predicted terpineol as the most compatible with cellulose diacetate, followed by methyl salicylate and citronellol. This was consistent with the plasticization efficiency trend from DMA results of solvent cast cellulose diacetate films containing the active compounds. The chemical identity of the films and cellulose diacetate-active compound intermolecular interactions were verified by FTIR. TGA demonstrated the thermal stability of cellulose diacetate/active compound/clay formulations at temperatures not exceeding 170 °C. Cellulose diacetate/organoclay nanocomposite matrices containing the active compounds at 35 wt-% were prepared by twin screw extrusion compounding, with the active compounds also functioning as plasticizers. The amount of active compound in the strands was determined by solvent extraction and TGA. Both methods showed that small amounts of active compound were lost during the compounding process. SEM demonstrated the effect of organoclay on the internal morphology of the matrix, whereas TEM showed clay dispersion and intercalation within the matrix. The matrix served as a reservoir for the active compounds while simultaneously controlling their release into the environment. Release profiles, obtained through oven ageing at 40 °C for 70 days, were fitted to existing Log-logistic and Weibull models, and novel Diffusion and Modified Weibull release models. Citronellol was released the fastest, followed by methyl salicylate and terpineol. The findings suggest that cellulose diacetate/organoclay strands are promising controlled-release matrices for pest control purposes.

Deep eutectic solvents aid the formulation of solid pesticide dosage forms for water-insoluble actives. This was demonstrated by encapsulating Amitraz powder in a low-melting matrix based on the eutectic mixture of urea (32 wt%) and 1,3-dimethylurea. Dissolution in water of melt-cast discs, containing 20 wt% active, led to the rapid release of Amitraz in a finely dispersed form. The order of magnitude reduction in particle size, after dissolution, is ascribed to the solubilization of Amitraz in the hot deep eutectic solvent and its subsequent precipitation as a separate phase on crystallization of the matrix.

The severity of tuberculosis (TB) in children is considered a global crisis compounded by the scarcity of pharmaceutical formulations suitable for pediatric use. The purpose of this study was to optimally develop and evaluate a pyrazinamide containing edible orodispersible film formulation potentially suitable for use in pediatrics actively infected with TB. The formulation was prepared employing aqueous-particulate blending and solvent casting methods facilitated by a high performance Box Behnken experimental design template. The optimized orodispersible formulation was mechanically robust, flexible, easy to handle, exhibited rapid disintegration with initial matrix collapse occurring under 60 s (0.58 &plusmn, 0.05 min &equiv, 34.98 &plusmn, 3.00 s) and pyrazinamide release was controlled by anomalous diffusion coupled with matrix disintegration and erosion mechanisms. It was microporous in nature, light weight (57.5 &plusmn, 0.5 mg) with an average diameter of 10.5 mm and uniformly distributed pyrazinamide load of 101.13 &plusmn, 2.03 %w/w. The formulation was physicochemically stable with no evidence of destructive drug&ndash, excipient interactions founded on outcomes of characterization and environmental stability investigations. Preliminary inquiries revealed that the orodispersible formulation was cytobiocompatible, palatable and remained intact under specific storage conditions. Summarily, an edible pyrazinamide containing orodispersible film formulation was optimally designed to potentially improve TB pharmacotherapy in children, particularly the under 5 year olds.

Safe application of water-insoluble acaricides requires fast release from solid dosage systems into aquatic environments. Dextrin is a water-soluble form of partially hydrolyzed starch, which may be used as matrix material for these systems if retrogradation can be inhibited by the inclusion of nanofillers. Several glycerol-plasticized thermoplastic dextrin-based nanocomposites were prepared with a twin-screw extrusion-compounding process. The nanofillers included a layered double hydroxide (LDH), cellulose nanofibers (CNF), and stearic acid. The time-dependent retrogradation of the compounds was monitored by X-ray diffraction (XRD) and dynamic mechanical thermal analysis (DMA). XRD showed that composite samples that included stearic acid in the formulation led to the formation of an amylose-lipid complex and a stable crystallinity during aging. The most promising nanocomposite included both stearic acid and CNF. It was selected as the carrier material for the water-insoluble acaricide Amitraz. Fast release rates were observed for composites containing 5, 10, and 20% (w/w) of the pesticide. A significant reduction in the particle size of the released Amitraz powder was observed, which is ascribed to the high-temperature compounding procedure.

The study investigated the toxicity effects of ‘form specific’ engineered nanomaterials (ENMs) and ions released from nano-enabled products (NEPs), namely sunscreens, sanitisers, body creams and socks on Pseudokirchneriella subcapitata, Spirodela polyrhiza, and Daphnia magna. Additionally, risk estimation emanating from the exposures was undertaken. The ENMs and the ions released from the products both contributed to the effects to varying extents, with neither being a uniform principal toxicity agent across the exposures, however, the effects were either synergistic or antagonistic. D. magna and S. polyrhiza were the most sensitive and least sensitive test organisms, respectively. The most toxic effects were from ENMs and ions released from sanitisers and sunscreens, whereas body creams and sock counterparts caused negligible effects. The internalisation of the ENMs from the sunscreens could not be established, only adsorption on the biota was evident. It was established that ENMs and ions released from products pose no imminent risk to ecosystems, instead, small to significant adverse effects are expected in the worst-case exposure scenario. The study demonstrates that while ENMs from products may not be considered to pose an imminent risk, increasing nanotechnology commercialization may increase their environmental exposure and risk potential, therefore, priority exposure cases need to be examined.

During melt extrusion of nanocomposites, nanoparticles can be incorporated either directly or through the masterbatch, which brings up a simple question of how the mode of nanoparticle inclusion affects the morphology and hence the rheological properties of the final composite. In the study reported here, nylon6/ethyl–vinyl-alcohol (N6/EVOH) was selected to model a blend system, and the effect of the mode of organoclay inclusion on the morphology development and melt-state viscoelastic properties of ternary blend composites was investigated. The morphological study using scanning electron microscopy, transmission electron microscopy, three-dimensional tomography, and differential scanning calorimetry indicated that the intercalated silicate layers were located in the interphase region between N6 and EVOH in the N6/EVOH/masterbatch composite and core–shell particles were formed, with EVOH as the core. On the other hand, the intercalated silicate layers were well distributed in the blend matrix of the N6/EVOH/organoclay composite, and it was difficult to differentiate between two phases. Finally, the different morphological developments were correlated with the melt-state rheological properties of the composites to evaluate the processing and product performance. The results showed that the masterbatch played an important role in establishing the compatibility of N6 and EVOH, and it could impede the gel formation that would benefit processing and product development.

In order to address risks associated with nanotechnology, there needs to be improved understanding of potential sources of engineered nanomaterials (ENMs) into the environment. Nano-enabled products (NEPs) are amongst such sources, and the current study identified NEPs sold in South Africa as a means of examining their potential to emit ENMs into the environment. Two hundred and sixty-four products were identified, mainly from the health and fitness category (66.7%), followed by electronics and computers (15.2%), home and garden (10.6%), appliances (3.8%), automotive (2.7%) and food and beverage category (1.1%). The chemical identity of ENMs was reported in 62.8% of those products with the top five most common being ENMs (in the descending order) Ti, C, Ag, Si and bisoctrizole. Determining the NEPs environmental exposure potential indicated that 63.6% exhibited high exposure potential comprising predominantly health and fitness, personal care, cosmetics and clothing products. The priority NEPs in terms of environmental exposure were sunscreens, hair relaxers, moisturising lotions, eye creams, cosmetic sera and socks. Assessed through analytical examination, NEPs were found to contain ENMs with length and width ranges of 15–350 and 8–185 nm, respectively. Our study suggests that most NEPs sold in South Africa pose a high potential for nanopollution, with water resources as the highest risk. Mandatory labelling of NEPs is recommended in order to better estimate the extent of nanopollution arising from usage of the NEPs.

Metal stearate blends are potential lubricants and filler dispersants in polymer compounding. Therefore, the thermal phase behaviour of binary blends of calcium stearate and magnesium stearate with zinc stearate was investigated. Cooling curve studies indicated lower melting temperatures in both systems. X-ray diffraction studies revealed that this was due to the formation of solid solutions of variable composition rather than to eutectic formation. Rheology measurements showed that true fluidification only happened well above the apparent melting points established from cooling curves. Calcium carbonate filled polyethylene containing metal stearate mixtures showed lower melt viscosities than were achievable using only calcium- or magnesium stearate as the lubricant. These results suggest that metal stearate blends, in combination with a judiciously selected wax component, may offer advantages in lowering compound melt viscosity and facilitating better particle dispersion.

Quaternary imidazolium salts with varying alkyl chain lengths (octyl, decyl, dodecyl, tetradecyl, hexadecyl) were synthesized and characterized with nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. Antibacterial screening experiments revealed that the activity decreased with increasing alkyl chain length. Quaternary imidazolium salts with alkyl chains, such as octyl and decyl, were used for the modification of montmorillonite (Mt). The characterization of the modified Mts was performed using XRD, TGA and TEM, and results showed that the surfactants were successfully intercalated in the Mt interlayer space. The selected quaternary imidazolium salt-modified Mts were evaluated for water disinfection using distilled, borehole and river water inoculated with Escherichia coli . Mt modified with surfactants carrying the octyl chains displayed excellent disinfection properties for all three water types. The TEM results showed that the mechanism of inactivation occurred through rupturing of the cell membrane after the E. coli cells came into contact with the modified Mt. In summary, the quaternary imidazolium-modified Mt has great potential to be used for household water disinfection.

Polylactide/butylated-starch/nanoclay (70/25.5/4.5 wt%) composites were prepared by melt blending with nanoclays of varying hydrophobicity. Electron microscopy studies indicated that the interphase boundary interaction was highest in the clay with intermediate hydrophobicity but decreased with clays of higher or lower hydrophobicity. Conventional and modulated differential scanning calorimetry studies showed that, in the case of composites, the change in the ratio of rigid and mobile amorphous fractions depends on the hydrophobicity of clays used for the preparation of blend composites. The mobile amorphous fraction related to the polylactide phase decreases in the case of intermediate hydrophobic clay compared with less and more hydrophobic clays or neat blend samples. Avrami bulk crystallization analysis and polarized optical microscopic observation demonstrated that hydrophobic clays hindered the crystallization of the PLA phase into the formation of disc-shaped spherulites. On the basis of obtained results, we propose a general understanding on how the morphology and thermal properties of the blend composites are related to the hydrophobicity of the nanoclays.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology.

In this article, a combined experimental and theoretical approach has been proposed to establish a relationship between the required shear force and the degree of delamination of clay tactoids during the melt-processing of polymer nanocomposites. Polypropylene (PP) was selected as a model polymer, and nanocomposites of PP with organically modified clay were prepared by a master batch dilution technique in a twin-screw extruder. The effect of PP throughput during the dilution of the master batch on the dispersion and orientation of clay platelets were studied in detail. Powder X-ray diffraction, small and wide angle X-ray scattering and high resolution transmission electron microscopy were used to study the structure and morphology of the obtained nanocomposites. The results showed that a lower feeding rate led to the orientation of clay platelets almost in the direction of extrusion. The adhesive force and the interaction energy between the clay platelets were theoretically calculated using the Hamaker approach. The analysis showed that the peeling mechanism is a practical explanation for the delamination of clay platelets during melt extrusion and that the dimensions of the clay platelet tactoids play an important role in the peeling due to the shear stress.

Copyright: 2014 Elsevier. This is an ABSTRACT ONLY. The definitive version is published in Applied Surface Science,vol. 311, pp 14-26

The combination of polymeric nanoparticles (NPs) as a core and lipid vesicles as a shell has emerged to be a robust and promising drug delivery strategy. This study explores the development of a novel combined delivery system where poly d,l, lactic-co-glycolic acid (PLGA) NPs are entrapped within Pheroid® drug delivery system. The solid NPs were combined with the Pheroid® vesicles using two different methods: pre-mix and post-mix. The surface properties of the PLGA NPs were altered through the inclusion (pos-NPs) and exclusion (neg-NPs) of chitosan (CT) and polyethylene glycol (PEG), to evaluate their interaction with the Pheroid® Vesicles. The average particle size of the novel NP–Pheroid® combined system ranged from approximately 1990–2450 nm while the zeta potential (ZP) ranged from −18 to −30 mV, measured using dynamic light scattering (DLS) and electrophoretic velocity techniques, respectively. The NP/Pheroid® mixing ratio experiment indicated that a maximum of 2.5% (w/v) NPs can be optimally added to the Pheroid® vesicles without compromising the structure and the stability of the NP–Pheroid® combined system. Visual analysis of this system was done through transmission electron microscopy (TEM), cryogenic (cryo) TEM and confocal laser scanning microscopy (CLSM) techniques to obtain adequate information of this novel combined drug delivery system which includes the localization of the PLGA NPs with the Pheroid ® vesicles.

The buccal mucosal route offers several advantages but the delivery of certain drugs can be limited by low membrane permeability. This study investigated the buccal permeability properties of didanosine (ddI) and assessed the potential of Aloe vera gel (AVgel) as a novel buccal permeation enhancer. Permeation studies were performed using Franz diffusion cells, and the drug was quantified by UV spectroscopy. Histomorphological evaluations were undertaken using light and transmission electron microscopy. The permeability of ddI was concentration-dependent, and it did not have any adverse effects on the buccal mucosae. A linear relationship (R² = 0.9557) between the concentrations and flux indicated passive diffusion as the mechanism of drug transport. AVgel at concentrations of 0.25 to 2 %w/v enhanced ddI permeability with enhancement ratios from 5.09 (0.25 %w/v) to 11.78 (2 %w/v) but decreased permeability at 4 and 6 %w/v. Ultrastructural analysis of the buccal mucosae treated with phosphate buffer saline pH 7.4 (PBS), ddI/PBS, and ddI/PBS/AVgel 0.5 %w/v showed cells with normal plasmalemma, well-developed cristae, and nuclei with regular nuclear envelopes. However, cells from 1, 2, and 6 %w/v AVgel-treated mucosae showed irregular nuclear outlines, increased intercellular spacing, and plasmalemma crenulations. This study demonstrates the potential of AVgel as a buccal permeation enhancer for ddI to improve anti-HIV and AIDS therapy.

South Africa has a rich flora which exhibits among the highest species density in the world, distributed across nine biomes that support an impressive diversity of animal life. However, a variety of human actions, invasion by alien species, natural disturbances and climate change collectively impact negatively on the great diversity of both plant and animal species. In situ conservation has long been practised, primarily in nature reserves, complemented by ex situ conservation in national botanic gardens, but in vitro plant conservation is not common. In the context of animal biodiversity conservation, the Wildlife Biological Resource Centre of the National Zoological Gardens utilises cryobanking as one of its major focuses and is now poised to expand as the repository for the cryoconservation of plant germplasm, particularly for indigenous recalcitrant-seeded and poor-seeding species. However, there are particular problems associated with successful germplasm cryostorage of such tropical and subtropical plants. As we see the science and application of cryobiology and cryoconservation as cross-cutting and transdisciplinary, we have entrained formal networking among scientists offering a range of specialisations aimed at a deeper understanding of common problems and practical outcomes to facilitate both plant and animal biobanking. The endeavours are aimed at elucidating the basis of both successes and failures in our efforts to attain optimal outcomes. With focus on best practices, standard operating procedures, validation and risk management for cryopreserved and cold-stored plant and animal material, our ultimate aim is to facilitate restoration by the safe reintroduction of indigenous species.

By designing and coupling two functional peptides, CKAFKRK and C(KAFKRK)3 in differing ratios to the surface of gold nanoparticles (GNPs), we evaluated the effect of loading on aggregation and proteolysis. Transmission electron microscopy images of the functionalised GNPs indicated a direct relationship between the degree of aggregation of the particles and the extent of peptide loading: The greater the percentage of the C(KAFKRK)3 peptide, the greater the dispersion (less aggregation) of the peptide-capped GNPs. The functionalised GNPs were subjected to trypsin digestion over increasing time periods and it was found that the peptides were cleaved at the site of Lys and Arg. The extent of cleavage was analysed by mass spectrometry. The results indicate that the rate of enzymatic degradation was directly proportional to the extent of loading, such that the greater percentage of the C(KAFKRK)3 peptide, the greater the rate and efficiency of the cleavage. These results could be attributed to the different peptide distribution of the particles and the entropy of the peptides with varying peptide ratios.

Heterocyclic cadmium dithiocarbamates, [Cd(S2CNC5H10)2] and [Cd(S2CNC9H10)2] were synthesized. The complexes were thermolysed in hexadecylamine (HDA) to give HDA capped CdS nanoparticles. A combination of close to spherical, rod, bipods and tripods was obtained by varying the reaction parameters such as precursor concentration and temperature. The optical properties and X-ray diffraction studies of the particles are also reported.

An increase in moisture during printing can lead to movement of fibres, such as puffing (return of a fibre to its uncollapsed state), twisting, and lifting of fibres out of the paper web. Spruce fibres are especially subject to these movements, contributing to weak bonding and increased surface roughness. The aim of the present study was to treat spruce pulp with enzymes to improve fibre bonding and stability. Spruce chemi-thermo-mechanical pulp was prepared in the laboratory and treated with mannanase, endoglucanase, and with a combination of these enzymes. Strength properties were evaluated before and after wetting of handsheets and their transverse sections were examined by scanning electron microscopy. Handsheet properties before and after rewetting as well as measurement of lumen area of fibres proved to be effective in evaluating fibre movement and puffing. The treatment of pulp with mannanase or endoglucanase improved fibre stability; however, endoglucanase treatment resulted in a loss of strength. Mannanase appeared to modify the cell walls, making them more susceptible to beating and resulting in improved fibre development and inter-fibre bonding. Endoglucanase possibly damaged fibrils, and fibre degradation instead of fibre development occurred during beating. Stability of thin-walled fibres improved more than thick-walled fibres after enzyme treatment.

The ultrastructure of dermal fibres of a 200 Myr thunniform ichthyosaur, Ichthyosaurus , specifically the 67 nm axial repeat D -banding of the fibrils, which characterizes collagen, is presented for the first time by means of scanning electron microscopy (SEM) analysis. The fragment of material investigated is part of previously described fossilized skin comprising an architecture of layers of oppositely oriented fibre bundles. The wider implication, as indicated by the extraordinary quality of preservation, is the robustness of the collagen molecule at the ultrastructural level, which presumably contributed to its survival during the initial processes of decomposition prior to mineralization. Investigation of the elemental composition of the sample by SEM–energy dispersive X-ray spectroscopy indicates that calcite and phosphate played important roles in the rapid mineralization and fine replication of the collagen fibres and fibrils. The exceedingly small sample used in the investigation and high level of information achieved indicate the potential for minimal damage to prized museum specimens; for example, ultrastructural investigations by SEM may be used to help resolve highly contentious questions, for example, ‘protofeathers’ in the Chinese dinosaurs.

To improve traditional cancer therapies, we synthesized polylactide (PLA) spheres coencapsulating magnetic nanoparticles (MNPs, Fe3O4) and an anticancer drug (doxorubicin, DOX). The synthesis process involves the preparation of Fe3O4 NPs by a coprecipitation method and then PLA/DOX/Fe3O4 spheres using the solvent evaporation (oil-in-water) technique. The Fe3O4 NPs were coated with oleic acid to improve their hydrophobicity and biocompatibility for medical applications. The structure, morphology and properties of the MNPs and PLA/DOX/Fe3O4 spheres were studied using various techniques, such as FTIR, SEM, TEM, TGA, VSM, UV-vis spectroscopy, and zeta potential measurements. The in vitro DOX release from the spheres was prolonged, sustained, and pH-dependent and fit a zero-order kinetics model and an anomalous mechanism. Interestingly, the spheres did not show a DOX burst effect, ensuring the minimal exposure of the healthy cells and an increased drug payload at the tumor site. The pronounced biocompatibility of the PLA/DOX/Fe3O4 spheres with HeLa cells was proven by a WST assay. In summary, the synthesized PLA/DOX/Fe3O4 spheres have the potential for magnetic targeting of tumor cells to transform conventional methods.

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g−1 dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s−1 or programmed cooling at 3·3 °C s−1. Samples were thawed rapidly (177 °C s−1) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination. Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth. Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and

A study on cryopreservation of excised embryonic axes of fifteen species of the amaryllidaceae is reported. Embryonic axes that after flash-drying had a water content in the range 0.4 to 0.1 g/g and survival greater than 60% were selected for cryopreservation procedures. The highest post-thaw viabilities (roots and shoots produced) across all species were recorded for embryonic axes subjected to rapid rather than slow cooling. With rapid cooling and no cryoprotection, the highest post-thaw viabilities for the fifteen species investigated was 0% in one species; ranged between 10 and 35% for nine species; and between 45 and 55% for five species. With cryoprotection and rapid cooling the highest post-thaw viabilities for these fifteen species was 0% for one species; ranged between 15 and 35% for six species; and between 40 and 75% for eight species. The highest post-thaw survival in ten out of fifteen species was obtained for axes dried to between 0.24 +/- 0.06 and 0.14 +/- 0.08 g/g(-1) (and rapidly cooled). With only one exception (Strumaria discifera; 45%), post-thaw survival after slow cooling ranged between 10 and 30%. Survival after vitrification plus slow cooling was achieved for seven species but was never higher than post-thaw survival in non-cryoprotected, rapidly cooled axes. The results suggest that species within the same family can exhibit commonalities in terms of amenability to cryopreservation techniques but for maximum success, axis water content and cooling rate particularly, must be optimised for each species in the family independently.

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 in70 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.

The present study investigated the rate of temperature change within axes of Poncirus trifoliata during cooling and warming by various methods. Cooling rates ranged between 0.17 and 170 degree (C per second, and warming rates of 1.25 and 600 degree C per second were measured when axes were warmed at room temperature or in water at 40 degree C, respectively. Partial drying increased the cooling rate within axes in direct contact with the cryogen, but did not affect the cooling or warming rates within axes enclosed in a double layer of lightweight aluminium foil. The procedures described illustrate the orders of magnitude that separate extremes of the range of cooling or warming rates attained using methods commonly employed in cryopreservation studies. Quantifying these rates allows the relationship between cooling rate, water content and survival of hydrated embryonic axes to be explored.

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.