Investigations into the responses of axes of recalcitrant seeds to dehydration and cryopreservation.

Thesis (Ph.D.)-University of Natal, Durban, 2002.Achieving long-term storage of germplasm is critical for the conservation of plant biodiversity. Seed storage practices require that degradative reactions causing ageing be limited. By reducing the water content, cytoplasmic viscosity is increased to levels that minimise deteriorative reactions. Reducing the storage temperature additionally increases the storage lifespan by further reducing the rate at which such deleterious processes occur. Two broad categories of seeds can be distinguished based on their storage behaviour. Orthodox seeds are desiccation-tolerant; generally shed in the dry state and are metabolically quiescent. Such seeds are usually stored at low water contents (e.g. 5%), and their high cytoplasmic viscosity prevents freezing damage during cooling to subzero temperatures. On the other hand, desiccation-sensitive (recalcitrant) seeds do not undergo a maturation-drying phase, they are metabolically active at shedding, and sensitive to extreme or prolonged drying. Accordingly, recalcitrant seeds cannot be stored under conventional conditions because they do not survive drying to low water contents and are damaged by sub-zero temperatures, even when dried to the lowest water content tolerated. Therefore, procedures that facilitate harmless drying and cooling to low temperatures are required to achieve long-term storage of recalcitrant germplasm. Recalcitrant seeds that are dried rapidly can attain relatively lower water contents without injury. However, these seeds are usually large and this limits the drying rates that can be achieved even under favourable conditions. Isolating embryonic axes from the rest of the seed facilitates faster drying, and a consequent reduction in the water content at which damage occurs. In axes of many species, the level of drying attained before lethal desiccation damage occurs is sufficient to limit freeZing damage during cryogenic exposure and facilitate survival in vitro. However, many others are damaged when dried to water contents that preclude freezing, and also are killed if cooled to sub-zero temperatures at higher water contents. In such instances, the window of permissible water contents leading to survival may be small or nonexistent. A basic premise explored in this thesis is that by restricting the growth of intracellular ice crystals using increasingly rapid cooling rates, the range of permissible water contents can be widened, facilitating survival of axes at higher water contents. An overview of the problems associated with the long-term storage of recalcitrant germplasm, and the rationale behind such rapid cooling approach are presented in Chapter 1 of the present thesis. Subsequent chapters report investigations on the effects of variables required to dry and cryopreserve embryonic axes with minimum damage, in keeping with this approach. Collectively, those studies aimed at establishing a robust cryopreservation procedure for the conservation of recalcitrant germplasm with broad applicability across species. The approach presently adopted entailed manipulating the water content of excised axes using rapid drying to discrete water content ranges, and also using different methods to cool axes to cryogenic temperatures at various rates. The calorimetric properties of water in axes were investigated for Camellia sinensis (L.) O. Kuntze using differential scanning calorimetry (DSC). For all species, the effect of any drying or cooling treatment tested was determined by assessing the survival of axes in vitro, which provided the most reliable indicator of cellular damage. Additionally, the effects of different treatments upon the structural and functional integrity of axes were assessed using light and electron microscopy as well as measurement of electrolyte leakage. The studies undertaken are presented in a similar sequence to that in which they took place during the course of the experimental phase of this work. These are summarised below. Partial drying plays a pivotal role in the approach developed, and microscopy has contributed towards increasing present understanding of desiccation damage. Microscopy was used to determine the effects of drying rate upon the ultrastructure of recalcitrant axes. It was necessary to find reliable protocols to prepare specimens for light and electron microscopy that did not alter the architecture of the cells in the dry state. Freeze-substitution and conventional aqueous fixation were compared in Chapter 2 using variously dried material from three species. The results obtained revealed that an unacceptably high extent of artefactual rehydration occurs during aqueous fixation, and highlight the need for anhydrous processing of dehydrated samples. Significantly, that study also revealed that many cellular events commonly associated with desiccation damage (e.g. withdrawal, tearing and/or vesiculation of the plasmalemma) are not seen in freeze-substituted preparations, and are likely artefacts of aqueous fixation. Freeze-substitution was used subsequently (Chapter 3) to assess the effects of slow drying (2 - 3 days) or rapid drying (min) upon the survival of embryonic axes of jackfruit (Artocarpus heterophyllus Lamk.) Results confirmed the beneficial effects of rapid drying, and also provided insights into ultrastructural changes and probable causes underlying cellular damage that occur during a drying/rehydration cycle. Efforts subsequently focused on determining the effect of cooling rate upon survival of recalcitrant axes at various water contents. The study on embryonic axes of recalcitrant camellia sinensis (tea; Chapter 4) tested the hypothesis that rapid cooling facilitates survival of axes at higher water content by restricting the growth of ice crystals to within harmless dimensions. The presence of sharp peaks in DSC melting thermograms was indicative of decreased survival in vitro. These peaks were attributed to the melting of ice crystals sufficiently large to be detected by DSC as well as to cause lethal damage to axes. Increasing the cooling rate from 10°C min-1 to that attained by forcibly plunging naked axes into sub-cooled nitrogen increased the upper limit of water content facilitating survival in vitro from c. 0.4 to 1.1 - 1.6 g H20 g-1 (dry mass [dmb]). Subsequent studies tested whether a similar trend occurred in other recalcitrant species cooled under similar conditions. In order to investigate further the relationship between water content, cooling rate and survival it was necessary to achieve cooling rates reproducibly, and to quantify these reliably. The plunging device required to achieve rapid cooling, and instruments required to measure the cooling rates attained, are described in Chapter 5. That study investigated the effects of cryogen type, depth of plunge and plunging velocity on the cooling rates measured by thermocouples either bare or within tissues of similar size and water content as encountered in cryopreservation experiments. This plunger was used in subsequent studies to achieve the fastest cooling conditions tested. Favourable cooling conditions were selected, and the efficacy of this procedure to cryopreserve relatively large axes was tested (Chapter 6) using embryonic axes of horse chestnut (Aesculus hippocastanum L.) Axes at water contents above c. 0.75 g g-1 could not be cooled faster than c. 60°C S-1, but cooling rates of axes below this water content increased markedly with isopentane, and to a lesser extent with subcooled nitrogen. Axes were killed when cooled at water contents above 1.0 g g-1 but survived fully (albeit abnormally) when cooled in isopentane between 1.0 and 0.75 g g-1. Complete survival and increasingly normal development was attained at water contents below 0.75 g g-1, especially if isopentane was used. The study on horse chestnut axes emphasised that water content and cooling rate are co-dependent during non-equilibrium cooling. Accordingly, that study could not determine whether survival at lower water contents increased because of the corresponding increase in cooling rates measured, or because of the higher cytoplasmic viscosity that resulted from drying. That uncertainty was addressed by the study discussed in Chapter 7, using axes of the trifoliate orange (Poncirus trifoliata [L.] RAF.) That study investigated the effect of cytoplasmic viscosity upon survival of axes cooled and warmed at different rates. Survival and normal development was high at lower water contents, and seemingly independent of cooling rate at about 0.26 g g-1. At higher water contents the range of cooling rates facilitating survival became narrower and displaced towards higher cooling rates. This study revealed two conspicuous inconsistencies that questioned the beneficial effect of rapid cooling. Firstly, the fastest cooling rates did not necessarily facilitate the highest survival. Secondly, survival of fully hydrated axes was higher when cooled under conditions that encouraged - rather than restricted - the growth of intracellular ice crystals. These inconsistencies were explored further using embryonic axes of silver maple (Acer saccharinum L.). Freeze-fracture replicas and freeze-substitution techniques provided valuable insights into the way in which ice crystals were distributed in cells cooled using different methods at rates ranging between 3.3 and 97°C S-1. Extensive intracellular freezing was common to all treatments. Unexpectedly, fully hydrated axes not only survived cryogenic exposure, but many axes developed normally when cooled using the relatively slower methods (77 and 3.3°C S-1) if warming was rapid. The most conspicuous ultrastructural difference between plunge cooling and the relatively slower methods was the exclusion of ice from many intracellular compartments in the latter. It is possible that even the fastest warming cannot prevent serious cellular damage if ice crystals form within such 'critical' compartments. It is proposed that the intracellular location of ice is a stronger determinant of survival that the size attained by ice crystals. The study of A. saccharinum also investigated the recovery of axes cooled fully hydrated either rapidly (97°C S-1) or slowly (3.3°C S-1). This facet of the study showed that cell lysis became apparent immediately after warming only where damage was most extensive. In other cells damage became apparent only after 2.5 to 6 h had elapsed, thus cautioning against inferring survival from the ultrastructural appearance of cells immediately after warming. Microscopy enabled cell repair as well as the pattern of growth of cryopreserved tissues to be appraised at the cellular, tissue and organ levels. Similar studies are required to understand further the nature of freezing damage, and how those events affect cell function. The salient trends observed in previous chapters are brought together in Chapter 9

Formulating calcium carbonate masterbatches

This study is based on the Master’s thesis of Mr. Radebe, supervised under the guidance of the other authors.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.Sasol.https://www.degruyter.com/journal/key/polyeng/html2023-11-07hj2023Chemical Engineerin

Characterizing CaCO3 particle dispersion in blown film

This study is based on the Master’s thesis of Mr. Radebe, supervised under the guidance of the other authors.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.Sasol, South Africa.https://www.degruyter.com/journal/key/polyeng/htmlhj2023Chemical Engineerin

Deep eutectic solvents for solid pesticide dosage forms

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 Institute of Applied Materials, University of Pretoriahttp://www.nature.com/srepam2021Chemical Engineerin

Cellulose acetate/organoclay nanocomposites as controlled release matrices for pest control applications

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.he Paper Manufacturers Association of South Africa (PAMSA) and the Department of Science and Innovation (DSI) and the National Research Foundation of South Africa (NRF).https://link.springer.com/journal/10570hj2023Chemical EngineeringUP Centre for Sustainable Malaria Control (UP CSMC

Morphology, thermal properties and crystallization kinetics of ternary blends of the polylactide and starch biopolymers and nanoclay : the role of nanoclay hydrophobicity

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.Department of Science and Technology (HGERAOZ) , the Council for Scientific and Industrial Research and the University of Johannesburg (086310).http://www.elsevier.com/locate/polymer2016-08-31hb201

Blooming of chlorfenapyr from polyethylene films

FTIR, in ATR and in transmission modes, enabled the tracking of the migration of the insecticide Chlorfenapyr to the surface of a polymer film. It also allowed estimation of the apparent solubility in the polymer. However, SEM investigations revealed that a portion of the insecticide is trapped, in a crystalline state, inside the polymer matrix.https://aip.scitation.org/journal/apcpm2021Chemical EngineeringUP Centre for Sustainable Malaria Control (UP CSMC

The Los Alamos Supernova Light Curve Project: Computational Methods

We have entered the era of explosive transient astronomy, in which upcoming real-time surveys like the Large Synoptic Survey Telescope (LSST), the Palomar Transient Factory (PTF) and Panoramic Survey Telescope and Rapid Response System (Pan-STARRS) will detect supernovae in unprecedented numbers. Future telescopes such as the James Webb Space Telescope may discover supernovae from the earliest stars in the universe and reveal their masses. The observational signatures of these astrophysical transients are the key to unveiling their central engines, the environments in which they occur, and to what precision they will pinpoint cosmic acceleration and the nature of dark energy. We present a new method for modeling supernova light curves and spectra with the radiation hydrodynamics code RAGE coupled with detailed monochromatic opacities in the SPECTRUM code. We include a suite of tests that demonstrate how the improved physics is indispensable to modeling shock breakout and light curves.Comment: 18 pages, 19 figures, published in ApJ Supplement