Cryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future.

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No description available.

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. / Thesis (Ph.D.)-University of Natal, Durban, 2002.

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.

The conservation of germplasm of indigenous plant species is vital not only to preserve valuable genotypes, but also the diversity represented by the gene pool. A complicating factor, however, is that a considerable number of species of tropical and sub-tropical origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated and metabolically active when shed and cannot be stored under conventional conditions of low temperature and low relative humidity. This poses major problems for the longterm conservation of the genetic resources of such species. Presently, the only strategy available for the long-term conservation of species that produce recalcitrant seeds is cryopreservation. Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The aim of the present study was to develop methods for the cryopreservation of germplasm of this species. Different explant types were investigated for this purpose, viz. embryonic axes (with attached cotyledonary segments) excised from seeds, and two in vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings and adventitious shoots generated from intact in vitro-germinated roots. Suitable micropropagation protocols were developed for all explant types prior to any other experimentation. Before explants could be cryopreserved it was necessary to reduce their water content in order to limit damaging ice crystallisation upon cooling. All explants tolerated dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with survival ranging from 100 – 80%, depending on the explant. In addition, penetrating and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants. The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose and glycerol. Explant survival following cryoprotection and dehydration ranged from 100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic temperatures by cooling at different rates, since this factor is also known to affect the success of a cryopreservation protocol. The results showed that ‘broken’ buds could not tolerate cryogen exposure. This was likely to have been a consequence of the large size of explants and their originally highly hydrated condition. Adventitious shoots tolerated cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached cotyledonary segments were exposed to cryogenic temperatures. In contrast, root production from axes cooled in sub-cooled nitrogen remained high (67 – 87%). Adventitious shoots were subsequently induced on roots generated from cryopreserved axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated roots. In this manner, the goal of seedling establishment from cryopreserved axes was attained. Each stage of a cryopreservation protocol imposes stresses that may limit success. To gain a better understanding of these processes the basis of damage was investigated by assessing the extracellular production of the reactive oxygen species (superoxide) at each stage of the protocol, as current thinking is that this is a primary stress or injury response. The results suggested that superoxide could not be identified as the ROS responsible for lack of onwards development during the cryopreparative stages or following cryogen exposure. The stresses imposed by the various stages of a cryopreservation protocol may affect the integrity of germplasm. Since the aim of a conservation programme is to maintain genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain whether this has been achieved. Thus, explants (axes with cotyledonary segments and adventitious shoots) were subjected to each stage of the cryopreservation protocol and the epigenetic integrity was assessed by coupled restriction enzyme digestion and random amplification of DNA. The results revealed little, if any, DNA methylation changes in response to the cryopreparative stages or following cryogen exposure. Overall, the results of this study provided a better understanding of the responses of germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant types were successfully cryopreserved. Future work can be directed towards elucidating the basis of damage incurred so that more effective protocols can be developed. Assessment of the integrity of DNA will give an indication as to the suitability of developed protocols, or where changes should be made to preserve the genetic (and epigenetic) integrity of germplasm. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Embryogenic tissue of Pinus patula Scheide et Deppe was initiated from immature green female cones during the months of November 1996 to February 1997 and December 1997 to January 1998. Tissue was maintained on MSG3 medium (BECWAR, NAGMANI and WANN 1990) supplemented with maltose. A comparison of various sugars as a carbohydrate source for maintaining embryogenic tissue showed that maltose was far superior to sucrose and the other sugars tested. Embryogenic tissue was successfully cryopreserved for up to 8 weeks using 0.3 M sorbitol and 5 % DMSO. Recovered tissue initially underwent a lag phase in tissue regrowth, but by the end of 5 weeks post-thaw, tissue proliferation was as vigorous as the unfrozen, untreated control. Fluoresceine diacetate (FDA) staining revealed that the embryonal head survived cryopreservation, but the highly vacuolated suspensor tissue had ruptured and died. Embryogenic tissue from two different families and four genotypes were successfully cryopreserved using this protocol. A comparison of commonly used cryopreservation techniques was conducted. It was found that the slow addition of the cryoprotectants over two days slowed the recovery rate of the tissue and increased the chances of contamination. Embryogenic tissue did not respond well to cryopreservation using a combination of the cryoprotectants PEG, glucose and DMSO (10-8-10%). Only a small proportion of the tissue survived, and initial tissue regrowth took up to 5 weeks. Embryogenic tissue was also set in gel and immersed directly in liquid nitrogen in an effort to cryopreserve tissue using the process of vitrification. However, none of the tissue survived, possibly due to insufficient dehydration prior to immersion in liquid nitrogen. Tissue recovery was highest when the tissue was precooled to -70°C in a container filled with isopropyl alcohol placed in a static freezer prior to immersion in liquid nitrogen. Recovery of tissue was improved by suspending the tissue on polyester grids and removing the liquid medium prior to placing onto MSG3 medium. Recovered tissue was bulked up using suspension cultures, and then paced onto MSG5 (BECWAR, NAGMANI and WANN 1990) or 240 medium (PULLMAN and WEBB 1994) to mature. Mature embryos were isolated from both media and germinated. Somatic plantlets were successfully hardened-off under greenhouse conditions. The successful cryopreservation of a number of genotypes and lines, and the maturation of recovered tissue has been achieved. This technique is now being actively incorporated into P. patula somatic embryo research, enabling the long-term storage of juvenile reference tissue while field trials are carried out and evaluated. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.

Cryopreservation is the most promising method for the long-term conservation of germplasm of plants producing desiccation-sensitive seeds. While such seeds are generally termed recalcitrant in the context of conventional storage practices, the term ‘cryo-recalcitrant’ is used for germplasm which is not readily amenable to cryopreservation. Cryo procedures usually involve a sequential combination of steps which must be optimised to limit the stresses experienced by specimens, thus promoting their survival. The present contribution reports on the effects of some of the steps involved in cryopreservation on the survival of the embryos of the amaryllids, Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos, with the ultimate aim of developing a protocol(s) for the successful cryopreservation of the germplasm of these species. The main foci of the investigations were the effects of rapid (flash) drying, the use of the cryoprotectant additives, glycerol (5 & 10%) and DMSO (0.1 & 0.25%), and employment of different cooling rates on the zygotic embryos of the selected species, which are known to be recalcitrant as well as being cryo-recalcitrant. Furthermore, this study reports on attempts at improving the rapidity of dehydration during flash drying by applying a vacuum, and also of providing cathodic protection (via highly reducing cathodic water and/or direct exposure to a static {negatively-charged} cathodic field during flash drying) to the explants at various stages in the protocol. These techniques were employed in attempts to ameliorate the adverse effects of reactive oxygen species associated with stresses imposed by the procedures during the cryopreservation process. The embryos of Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos were initially at water contents (WCs, dry mass basis) of 3.28±0.52, 2.55±0.22, 4.48±0.92 g g-1, respectively, after harvest. These embryos proved to be tolerant to moderately rapid water loss in the short term, with >60% retaining germinability at water contents ≥0.5 g g-1. The results from this study confirmed that dehydration to water contents below 0.5 g g-1 (dry mass basis) compromised survival, and that this effect was exacerbated if the embryos were cryoprotected prior to drying. Interestingly, the rate of water loss in embryos of these species differed, with A. coranica and H. albiflos drying at a (comparably) much slower rate than those of B. grandiflora. Subsequent rapid cooling yielded promising results when compared with slow cooling, as 30% of glycerol cryoprotected, rapidly cooled A. coranica embryos that had been flash-dried to 0.36±0.10 g g-1 generated normal seedlings. It was clear, however, that the effects of these procedures were exacerbated when all the steps of the cryo procedure were applied sequentially. However, the work also showed that these adverse effects may be ameliorated if each step of the cryopreservation protocol is optimised on a species-specific basis, thus promoting the chances of survival after cryopreservation and facilitating subsequent seedling establishment. This was evident in the 30% germination obtained when embryos of A. coranica, which had been cryoprotected with glycerol prior to flash drying before exposure to rapid cooling, while those that had not been cryoprotected or were cryoprotected with DMSO before drying did not survive. The incorporation of cathodic protection during flash drying appeared promising as it promoted the survival of 10% of H. albiflos embryos dehydrated to WCs between 0.37 and 0.26 g g-1 (whereas no survival was achieved without the inclusion of this step), and 70% of A. coranica embryos that were dehydrated to 0.35±0.21. In addition, the reduction of the explant size, from a whole 6 mm embryo to a 3-4 mm excised axis, promoted survival by up to 30% for A. coranica and H. albiflos, even at higher WCs. However, survival in these cases was based on observations of abnormal development, i.e. the development of roots or shoots, or calli. No surviving embryos were obtained from B. grandiflora after cooling, regardless of the preconditioning treatment or rate of cooling, and this was accredited to the greater degree of sensitivity of these embryos to the cryo procedures than those of the other two species. The use of cathodic water to re-hydrate explants after dehydration and of applying a vacuum during flash drying did not result in any observable benefits, and require further investigation for optimisation. The very limited success towards establishing a cryopreservation protocol for the species investigated in this study reinforces the difficulties associated with the cryopreservation of recalcitrant germplasm, which informs the cryo-recalcitrance of some explants. However, the results obtained have helped to identify a number of intervention points that could be used to minimise the damage incurred during the various procedural steps involved in cryopreservation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.