Browsing by Author "Wesley-Smith, James."

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Investigations into the responses of axes of recalcitrant seeds to dehydration and cryopreservation.
(2002) Wesley-Smith, James.; Berjak, Patricia.; Walters, Christina.; Pammenter, Norman William.
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.
Optimising methods for embryonic axis fixation and micropropagation of Syzygium cordatum Hochst.
(2009) Premsagar, Varsha.; Berjak, Patricia.; Pammenter, Norman William.; Wesley-Smith, James.
Syzygium cordatum Hochst. (family - Myrtaceae), commonly called the umDoni (Zulu) tree, is found throughout South Africa. The tree is utilised for its fruit, bark and wood by many villagers, and this demand has placed potential pressure on existing populations. It is necessary to conserve this widely used tree before it becomes threatened by over-utilisation. Seeds of S. cordatum are recalcitrant and storable only in the short-term at 16oC over moist paper towel (hydrated storage). The study was initiated to follow deterioration of the embryonic axes, in relation to dehydrated versus and hydrated storage. However, for electron microscopic investigations, it was crucial that material was properly fixed to obtain samples that accurately represented the in vivo conditions. This proved to be challenging, as explained below, and changed the original aim of the project. The high phenolic content of S. cordatum seeds and axes makes fixation, using an aldehyde-based fixative, such as glutaraldehyde, difficult, as the aldehyde groups bind to phenolic compounds, forming large oligomers that tear out during sectioning. This causes sections to become fragmented, making viewing with the transmission electron microscope (TEM) impossible. The quest to visualise the ultrastructure, consequently became an additional focus of the project. Substituting glutaraldehyde with alternate primary fixatives including potassium permanganate (KMnO4) and 1% osmium tetroxide (OsO4) did not improve the situation. Cryo-fixation followed by freeze substitution was then attempted. Three substitution media, comprising glutaraldehyde, tannic acid, osmium tetroxide and acetone were used, all providing similar, unsatisfactory results showing ice crystal damage. Eventually, glutaraldehyde fixation was modified where samples were fixed in glutaraldehyde while being exposed to microwave energy. Results from this method of fixation were far better, with fine structure adequately preserved. A second facet of the project was aimed at producing explants alternative to seed-derived zygotic axes. Cotyledonary explants used in an attempt to produce somatic embryos, were cultured onto media which incorporated various concentrations of 2,4-D, BAP and NAA. The callus produced was sub-cultured onto regeneration media, which included NAA and BAP or PGR-free media, did not develop further. Zygotic axes cultured onto shoot multiplication medium containing BAP and NAA produced adventitious shoots which produced roots when sub-cultured onto media containing GA3.
Studies on factors influencing viability after cryopreservation of excised zygotic embryos from recalcitrant seeds of two amaryllid species.
(2010) Naidoo, Sershen.; Berjak, Patricia.; Pammenter, Norman William.; Wesley-Smith, James.
Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long periods in the dry state and hence can be stored under low relative humidity and temperature conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at moderate temperatures, allows for short- to medium-term storage but only facilitates viability retention for a matter of a few weeks to months, at best, because the seeds are metabolically active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning treatments can reduce the amount of ‘free’ water available for freezing and may increase the chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw survival in some recalcitrant-seeded species. However, there is a paucity of information on the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and this is the major focus of the current contribution. Additionally, in light of the lack of understanding on how cryo-related stresses imposed at the embryonic stage are translated or manifested during subsequent seedling growth, this study also investigated the effects of partial dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South Africa. Studies described in Chapter 2 aimed to interpret the interactive effects of partial dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative] or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic embryos, stresses and lesions, metabolic and physical, induced at each stage of the cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further damage and/or viability loss with the progression of each step. Maximum post-thaw viability retention in both species appeared to be based on the balance between desiccation damage and freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities in both species were best when Gly cryoprotected + partially dried zygotic embryos were rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed during rapid drying was higher in A. belladonna and this may explain why the optimum water content range for post-thaw survival was <0.40 g g-¹ for A. belladonna and >0.40 g g-¹ for H. montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to be the product of both the ‘intensity’ and ‘duration’ of the stress. Cryoprotection and dehydration increased the chances of post-thaw survival in A. belladonna and H. montanus zygotic embryos. However, transmission electron microscopy studies on the root meristematic cells from the radicals of these embryos (described in Chapter 3) suggest that their practical benefits appear to have been realised only when damage to the sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised both dehydration and freezing damage. The ability of A. belladonna and H. montanus embryos to tolerate the various components of cryopreservation in relation to changes in extracellular superoxide (.O2 -) production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in oxidative stress and the accompanying decline in viability suggests that oxidative stress was a major component of cryoinjury in the embryos presently investigated. Post-thaw viability retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected + partially dried embryos, possibly due to the relatively lower post-drying lipid peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant activities in the former. Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide (H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5) increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw total antioxidant and viability loss than untreated embryos. To assess the vigour of seedlings recovered from partially dried H. montanus embryos, seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic embryos during cryopreservation may compromise the vigour (e.g. development of persistent low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance of recovered seedlings, compared with seedlings recovered from fresh embryos. While the adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative growth rate of seedlings from partially dried embryos). The reduced vigour and drought tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present work may therefore disappear with an extension in the period afforded to them for hardening-off under green-house conditions, and in the field. The results presented in this thesis reinforce the notion that each successive manipulation involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict damage on tissues and post-thaw survival in such germplasm relies on the minimisation of structural and metabolic damage at each of the procedural steps involved in their cryopreservation. The results also highlight the need to design research programmes aimed not only at developing protocols for cryopreservation of plant genetic resources, but also at elucidating and understanding the fundamental basis of both successes and failures.