Doctoral Degrees (Botany)

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Desiccation tolerance and sensitivity of vegetative plant tissue.
(1995) Sherwin, Heather Wendy.; Pammenter, Norman William.; Berjak, Patricia.
There is a great deal of work currently being done in the field of desiccation tolerance. Generally workers studying desiccation-tolerant plant tissues have concentrated on the mechanisms of desiccation tolerance without concomitant studies on why most plants cannot survive desiccation. The present study considers both a desiccation-tolerant plant as well as a range of desiccation-sensitive plants. The work incorporates physiological, biophysical, biochemical and ultrastructural studies in an attempt to get a holistic picture of vegetative material as it dries and then rehydrates. The plant species used in this study are: Craterostigma nanum, a so-called resurrection plant; Garcinia livingstonei, a drought-tolerant small tree; Isoglossa woodii, an understorey shrub which shows a remarkable ability to recover from wilting; Pisum sativum seedlings, which have a very high water content at full turgor; and finally, Adiantum raddianum, the maiden hair fern, which wilts very quickly and does not recover from wilting. The desiccation-tolerant plant, C. nanum, had an unusual pressure-volume (PV) curve which indicated that while large volume changes were taking place there was little concomitant change in pressure or water potential. The unusual nature of this PV curve made it difficult to assess the relative water content (RWC) at which turgor was lost. The desiccation-sensitive plants exhibited standard curvi-linear PV curves. The amount of nonfreezable water in the five species was studied and found to show no correlation with the ability to withstand dehydration or with the lethal water content. There were no differences in the melting enthalpy of tissue water between the tolerant and most of the sensitive plants. Isoglossa woodii had a lower melting enthalpy than the tolerant and the other sensitive species. Survival studies showed that the desiccation-sensitive plants all had similar lethal RWCs. The tolerant plant survived dehydration to as low as 1% RWC, recovering on rehydration within 24 hours. Membrane leakage studies showed that the sensitive plants all exhibited membrane damage at different absolute water contents, but very similar RWCs and water potentials. The increase in leakage corresponded to the lethal RWC for all the sensitive species. The desiccation-tolerant plant recovered from dehydration to very low water contents and did not show an increase in membrane leakage if prior rehydration had taken place. Without prior rehydration this tolerant plant exhibited an increase in leakage at similar RWCs and water potentials to that of the sensitive species. There did not appear to be much difference in the RWC at which damage to membranes occurred whether the material was dried rapidly or slowly. Respiration and chlorophyll fluorescence were studied to determine what effect drying and rehydration have on the electron transport· processes of the leaf. The chlorophyll fluorescence studies gave an indication of damage to the photosynthetic apparatus. Both qualitative changes as well as quantitative changes in fluorescence parameters were assessed. Characteristics like quantum efficiency (Fv/Fm)remained fairly constant for a wide range of RWCs until a critical RWC was reached where there was a sharp decline in Fv/Fm. Upon rehydration, C. nanum recovered to pre-stress levels, I. woodii showed no recovery and no further damage on rehydration, whilst the other species exhibited even more damage on rehydration than they had on dehydration. Respiration remained fairly constant or increased slightly during drying until a critical RWC was reached at which it suddenly declined. The RWC at which this decline occurred ranged from 15% and 20% in P. sativum and C. nanum respectively, to 50% for G. livingstonei. On rehydration respiration exceeded the levels measured in dehydrated material for the sensitive species. Unsuccessful attempts were made to fix material anhydrously for ultrastructural studies so standard fIxation was used. The ultrastructural studies revealed that changes had occurred in the ultrastructure of leaves of the sensitive species dried to 30% RWC particularly in A. raddianum and P. sativum. Drying to 5% RWC revealed extensive ultrastructural degradation which was worsened on rehydration in the sensitive species. The tolerant species showed ultrastructural changes on drying but these were not as severe as occurred in the sensitive species. The cell walls of the tolerant species folded in on drying. This folding was possibly responsible for the unusual PV curves found in this species. At 5% RWC the cells were closely packed and very irregular in shape. The cell contents were clearly resolved and evenly spread throughout the cell. The large central vacuole appeared to have subdivided into a number of smaller vacuoles. On rehydration the cells regained their shape and the cell contents had moved towards the periphery as the large central vacuole was reformed. Beading of membranes, which was common in the sensitive species, was not found in the tolerant species suggesting that membrane damage was not as severe in the tolerant species. Western Blot analysis of the proteins present during drying was performed to determine whether a class of desiccation-induced proteins, called dehydrins, were present. These proteins have been suggested to play a protective role in desiccation-tolerant tissue. It was found that C. nanum did, in fact, possess dehydrins, but so did P. sativum. The other three sensitive species did not show any appreciable levels of dehydrin proteins. The presence of dehydrins alone is, therefore, not sufficient to confer desiccation tolerance. While physiologically the damage occurring in the sensitive plants was similar to that of the tolerant plant, at an ultrastructural level the damage appeared less in the tolerant plant. On rehydration from low RWCs damage appeared to become exacerbated in the sensitive plants. This was in contrast to the tolerant plant where damage was apparently repaired. There appears, therefore, to be a combination of protection and repair mechanisms responsible for the ability of C. nanum to tolerate desiccation. The lethal RWC of the sensitive species was higher than that at which protective mechanisms, such as water replacement, might come into play. So it is not just the possible ability to replace tightly bound water that set the tolerant plant aside. It must also have mechanisms to tolerate damage at the higher RWCs which were damaging and lethal to the sensitive plants. The lethal damage to sensitive species appeared to be related to a critical volume, thus it is concluded that the tolerant plant had the ability to tolerate or avoid this mechanical damage during drying as well as the ability to remain viable in the dry state. It is hypothesised that the ability of the walls to fold in and the unusual nature of the PV curve may provide some answers to the enigma of desiccation tolerance.
Development of strategies towards the cryopreservation of germplasm of Ekebergia capensis Sparrm. : an indigenous species that produces recalcitrant seeds.
(2011) Hajari, Elliosha.; Berjak, Patricia.
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.
Effects of antifungal treatments on some recalcitrant seeds.
(2017) Makhathini, Aneliswa Phumzile.; Berjak, Patricia.; Pammenter, Norman William.
Abstract available in PDF file.
Endogenous and exogenous factors involved in sorghum germination with reference to malting.
(1997) Dewar, Janice.; Berjak, Patricia.; Taylor, John.
In Africa, the grain sorghum (Sorghum bicolor (L.) Moench), is malted to provide the most important ingredient in brewing, malt, which is used primarily for the production of traditional (opaque) sorghum beer. Malting is the germination of cereal grain in moist air under controlled conditions, the primary objective being to promote the development of hydrolytic enzymes which are not present in the ungerminated grain. The malting process can be physically split into three distinct unit operations (viz. steeping, germination and drying). To date, little attention has been given to optimising the conditions of steeping for sorghum. The effects of different steeping variables (time, temperature and aeration) on the quality (in terms of diastatic power (amylase activity), free amino nitrogen and hot water extract) of sorghum malt for brewing were investigated. Malt quality was found to increase with steeping time, over the range 16-40 hours and the optimum steeping temperature was found to be in the range 25 to 30°C. Aeration during steeping appeared to be necessary to maximise the malt quality, particularly when steeping was conducted for long periods at high temperatures. Of particular significance was the observation that final sorghum malt quality was highly significantly correlated (p<0.01) with grain moisture content at steep-out (the end of the imbibition period). When steeping conditions based on these findings were used, a germination temperature of 25-30°C was found to be optimal for sorghum malt quality. As with steep-out moisture, green malt (grain after the specified germination time) moisture content was correlated Significantly (p<0.01) with final sorghum malt quality. The finding that sorghum malt quality is related to steepout moisture content was given further substance when it was shown that the stimulatory effect on sorghum malt quality of steeping sorghum in a dilute solution of alkali, actually increases the amount of water taken up during steeping probably because the alkali disrupted the pericarp cell wall structure of the grain. Barley malting practices have taken advantage of the knowledge that the exogenous application of gibberellic acid can enhance the synthesis of the critically important malt hydrolytic enzyme, a-amylase. To date, literature on the effect of exogenous application of gibberellic acid on sorghum malt quality has been inconclusive; with reports both of no effects, and of positive effects, on amylase activity. To elucidate the possible control mechanisms involved in sorghum germination, a combined HPLC-radioimmunoassay technique was used to determine the levels of selected plant growth regulators from the groups auxin, cytokinins, gibberellins and abscisic acid in sorghum at various stages of germination. Levels of gibberellic acid were low throughout germination. During germination the levels of the other plant growth regulators declined, but a peak in cytokinins followed the first visible signs of root protrusion. The high level of the germination inhibitor and gibberellic acid antagonist, abscisic acid, in the germ (embryo inclusive of scutellum) portion of the mature non-germinated grains was noteworthy. Based on these findings, it was determined that sorghum malt quality could in fact be improved significantly by the application of exogenous gibberellic acid. However, this was effective only if it was administered during the end of steeping or at the beginning of the germination step. By optimising the conditions of steeping and germination and by steeping in dilute NaOH or in gibberellic acid not only should it be possible to enhance the quality of sorghum malt, it should be possible to reduce the time required to obtain the specific quality, thereby offering a saving to the sorghum maltster in terms of operation costs and enhancing the total throughput possible from the malting plant.
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.
Some implications of associated mycoflora during hydrated storage of recalcitrant seeds of Avicennia marina (Forssk.) Vierh.
(2004) Calistru, Claudia.; Berjak, Patricia.; Pammenter, Norman William.; McClean, Michelle.
Three questions are considered in the context of the possible effects of seedassociated mycoflora, typified by Fusarium moniliforme, during hydrated storage of recalcitrant seeds of the tropical species, Avicennia marina. These are: 1) whether fungal infection reduces storage lifespan; 2) whether seeds become more susceptible to fungal attack during storage and whether they posses defence mechanisms that might suppress fungal proliferation in hydrated storage (production of antifungal compounds and 13-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14)] and 3) whether it is possible to discriminate ultrastructurally between inherent deteriorative changes and those that are fungally-induced. 1) The data indicate unequivocally that if fungal activity is curtailed, then the hydrated storage lifespan of A. marina seeds can be considerably extended. 2) When inoculated immediately with F. moniliforme, newly harvested seeds were extremely susceptible to the adverse effects of the fungus, while seeds that had been wet-stored for 4 days showed a considerably heightened resilience to the effects of the fungus prior to inoculation. The enhanced resilience, although declining, persisted in seeds stored hydrated for up to 10 days prior to inoculation, being lost after 12 days. This finding was supported by significant increase in 13-1,3-glucanase and chitinase and in antifungal compound production during 10 days of wet storage. After 14 days of wetstorage, seeds become more susceptible to the effects of fungusthanthose in the newly harvested condition. 3) The resilience of seeds that had been stored in the short-term was associated with ultrastructural changes indicative of enhanced metabolic activity associated with the onset of germination (e.g. increase in vacuolation, well-developed mitochondria and endomembrane system [ER and Golgi bodies]). However, with sustained stress associated with wet-storage IV conditions, the seeds became increasingly badly affected by the fungus, showing some ultrastructural fungally-induced abnormalities (e.g. nuclear lobing, presence of lipid bodies and prevalence of Golgi bodies that had many associated vesicles) and a decrease in 13-1,3-glucanase and chitinase activity. It is suggested that the decreased susceptibility of A. marina seeds during short-term storage relies on the ability to create an antifungal environment prior to infection (through synthesis and accumulation of pre-formed and induced antifungal compounds and antifungal enzymes), which would also be an effective strategy during germination in the natural environment.
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.
Towards ameliorating some of the stresses associated with the procedural steps involved in the cryopreservation of recalcitrant-seeded germplasm.
(2017) Naidoo, Cassandra Dasanah.; Berjak, Patricia.; Pammenter, Norman William.; Varghese, Boby.
Abstract available in PDF file.

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