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LD2668 .T4 1947 H39 / Master of Science

The primary grain in oat spikelets is larger than the secondary grain. Oat genotypes were grown to determine the effects of planting primary and secondary grains separately on vegetative and reproductive growth. Plants from primary seeds had higher yields than those from secondary seeds. The advantage was associated with earliness of heading. It was concluded that increased seed uniformity would be advantageous. / Reductions in panicle size by removal of all primary grains and some secondary grains indicated that secondary grains have more growth potential than observed in untreated panicles. However, secondary grains in the treated panicles did not achieve weights equalling those of primary grains in the untreated panicles. / Variation in primary:secondary seed weight ratios was found among experiments. The variability was attributed mainly to instability of secondary grain weight. / The inheritance of secondary grain weight and of the primary:secondary seed weight ratio was studied at two locations. For both traits, additive and non-additive genetic effects were evident. Dominant gene action was relatively unimportant, suggesting good potential for improvement through pure-line breeding.

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004

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.

Dormancy characteristics and optimum conditions for germination of Cleome gynandra seeds have not been explained. Seed storage proteins were extracted, analysed with SDS-PAGE and sequenced. Seed proteins of Cleome were characterised by comparison with those of wild mustard (Brassica kaber). Wild mustard showed seed proteins composed of two α-chains of molecular weight (24-32 kDa) and another two β-chains of 18-22 kDa. The seed proteins of Cleome comprised two α-chain polypeptides of molecular weight (25-30 kDa), two β-chain polypeptides of molecular weight (18-20 kDa) and a smaller β-chain of 13-15 kDa. The storage proteins occurred in the seeds as dimeric complexes of molecular weight 40-65 kDa, which were broken into polypeptide chains of approximately 20 and 30 kDa by the reducing . action of DTT. Comparison with proteins in the proteome library and similarity index further confirmed that the seed proteins of Cleome had similarities with those of wild mustard. Two dimensional SDS-PAGE showed that the two species have nine similar polypeptides and four different ones. Events associated with dormancy release during seed germination still require explanation. Seeds of Cleome are characterised by low germination and there has been no explanation for this. Changes in protein expression during germination of Cleome in the presence or absence of light and at constant or alternating temperatures were examined. The germination of Cleome seeds at 20 degrees C was inhibited by light, but it was improved at 20 degrees C in darkness. There was no photoinhibition when seeds were germinated at constant 30 degrees C or alternating 20/30 degrees C (16 h night and 8 h day) for 10 days. Four proteins were observed to decrease in expression as germination progressed, but remained unchanged during photoinhibition. Photoinhibition was expressed more in seeds that were harvested late, after the pods had turned brown. These seeds showed a fifth, low molecular weight protein (13 kDa) that was absent from the immature seeds and embryos. Photinhibition is a pseudo-dormancy condition during which seed storage proteins are not utilised and the seed coat could partially play a role in it. The temperatures for the germination of Cleome in darkness have been determined. However, prior to this study the effects of temperature, light and pre-germination treatments (chilling, scarification, hydration and germination in the presence of KN0(3) or GA(3) on the germination of the seeds of this species have not been investigated. Seeds were germinated for 10 days and the final count of germination was used to determine seed performance. The highest germination percentage (60% and 80%, for a 2-year old and a l-year old seed lot, respectively) of untreated seeds was achieved when alternating temperatures of 20/30 degrees C (16 h/S h) in the dark or constant 30 degrees C in the dark were used. Among the pre-germination treatments, only scarification (puncturing of seeds at the radicle end) improved germination. Seeds were found to be negatively photoblastic, and the phenomenon was more pronounced when they were germinated at 20 degrees C and 12 h photoperiod or longer. Germination of photoinhibited seeds was, however, improved by treatment with GA(3) It is recommended that the germination of Cleome be undertaken under conditions of darkness and at either alternating 20/30 degrees C or continuous 30 degrees C. Seed lot vigour and seedling vigour are two important seed quality aspects that are used in defining the seed germination process. Seed germination is appropriately characterised by radicle protrusion and the attainment of normal seedling structures. However, the international rules for testing seeds combine radicle protrusion and normal seedling attainment in separating seed germination into the first and final counts. The challenge to a seed analyst testing the germination of a species whose first and final counts are unknown is that there is no statistical guideline to determine these important stages of seed germination. Cauliflower and broccoli, for which the first and final counts are published in the international rules for testing seeds and Cleome, for which there is no data on the first and final counts, were examined to determine the statistical significances of the first and final counts. Analysis of variance, logistic regression, 'broken-stick' regression models and survival analysis procedures were used. Analysis of variance showed that there were no differences between the germination percentages on the fourth, fifth and seventh days of germination. Low and stable standard deviations were recorded when evaluating germination after the fourth day. The germination curves of broccoli and cauliflower did not fit the Gompertz curve but fitted the exponential curve. The broken-stick model 'broke' the cumulative germination curve for the Cleome seed lots into two linear curves that were significantly different, but failed to break those for broccoli and cauliflower. However, this study confirmed the first and final counts for broccoli and cauliflower as determined by the international rules for testing seeds. Broken-stick modelling and life table analyses confirmed the fourth day as being appropriate to determine the first count for Cleome germination. There was no evidence of further seed germination after the seventh day as shown by probability density and hazard rate. It is suggested that for Cleome, the 'first count' and 'final count' be performed on the fourth and seventh day of the germination, respectively. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Seeds remain the most convenient and successful way for storing the genetic diversity of plant species and for producing new plants routinely for agriculture and horticulture. The importance of seed storage and the ability to predict seed longevity must therefore not be underestimated. To be successful, storage conditions must maintain seed vigour and viability and ensure that normal seedlings are subsequently established under field conditions. Seed quality is best retained when deteriorative events are minimised, which is achieved by storage of low moisture-content seeds under cool to cold, or even sub-zero, temperatures. Such conditions are employed for 'orthodox' seeds, which are desiccation tolerant and able to survive at sub-zero temperatures in the dehydrated state for extended periods. It is seeds referred to as 'recalcitrant' that cannot be dehydrated and often not stored at low temperatures because they are desiccation sensitive and may not tolerate chilling. According to almost anecdotal records chilling temperatures for such seeds are those below 15°C down to 0°C, depending on the species. The limited storage lifespan of recalcitrant seeds presents a problem even for short-term storage, and as most research on chilling sensitivity has been conducted on vegetative tissue, relatively little data exist for seeds, especially recalcitrant types. The purpose of this study was to gain an understanding of the chilling response of recalcitrant seeds, as reduced temperature could have the potential to extend, rather than curtail, storage lifespan, depending on the species. Selected physiological, biochemical and ultrastructural responses of recalcitrant seeds of Avicennia marina and Ekebergia capensis were characterised. Seeds of the two species were stored at 25, 16 and 6°C. Germination, water content (determined gravimetrically), respiration (measured as CO2 production) and leachate conductivity (tissue electrolyte leakage over time) were assessed at regular intervals. Chilling response at the subcellular level was examined using transmission electron microscopy (TEM). Changes in sugar metabolism and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were assessed for A. marina seeds, which were severely affected by the chilling temperature of 6°C, losing viability after 1 week. In contrast, the seeds of E. capensis retained viability after 12 weeks of storage at 6°C, indicating the marked difference in chilling response between seeds of the two recalcitrant species, despite their common tropical provenance. However, when E. capensis seeds were stored at 3°C viability decreased significantly after 8 weeks, thus indicating how critically temperature must be controlled if such conditions are to be profitably employed. Ultrastructural studies revealed that in both E. capensis and A. marina seeds vacuole formation was initiated more rapidly at lower temperatures than at higher temperatures, indicating that this was a response specific to the chilling stress imposed. Once again, 'lower temperatures' differed relative to the species concerned. In the E. capensis seeds, nucleolar morphology was affected and the extent of chromatin patches in the nuclei increased as the storage temperature was reduced. Other ultrastructural findings could not be linked specifically to the chilling stress imposed on the E. capensis and A. marina seeds. Activity of the antioxidant enzymes SOD and GR was detected in the A. marina seeds. No measurable CAT activity was detected. Glutathione reductase activity increased in response to chilling stress, the rate of the increase depending upon the severity of the chilling stress imposed. Other than when the A. marina seeds were placed directly at 6°C, there were no notable increases in SOD activity. Interestingly, SOD and GR activity was not the same in the axes as in the cotyledons. Superoxide dismutase activity was found to be higher in the axes and GR activity higher in the cotyledons. It would have been beneficial to determine the extent of antioxidant enzyme activity in the E. capensis seeds as well if this had been possible. Generally, chilling of recalcitrant seeds seems to evoke a response similar to that of dehydration below a critical water content. This could lead to the conclusion that recalcitrant seeds do not possess the genetic ability to cope with dehydration or chilling stress, if it were not for the existence of recalcitrant seed species that are more chilling tolerant. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

The consequences of toxigenic fungi associated with stored seed have stimulated these investigations aimed at developing treatments to minimise this mycoflora, without significantly reducing seed quality or viability. The effects of immersion in water at 55, 57 and 60 QC for durations of 5 to 60 min were assessed for maize (Zea mays L.) seed in terms of fungal status, water uptake, electrolyte leakage, germination and seedling establishment. These assessments were conducted immediately after treatment, after re-dehydration for 2 days in an ambient air stream, and following a 1 month storage period under either cold (4 QC) or ambient (25 QC) conditions (33% and 91% RH, respectively). In all cases, the results are compared with those of control seeds and seeds pre-imbibed for 4 h at ambient temperature. The level of internal contamination, represented almost entirely by Fusarium moniliforme Sheldon, declined significantly when assessed immediately after treatment, the efficacy of which increased with increasing temperature and duration of treatment. Seeds immersed in water at 55 QC for a duration of 15 min exhibited an 85% reduction in infection levels, when compared with those of the control, while those treated at 57 and 60 QC (same duration) were uninfected. Immersing seeds in hot water, however, resulted in a lag in germination rate and drop in germination totality, the degree of which was enhanced by increasing duration and temperature of treatment, suggesting the status of the manipulation to be an accelerated ageing treatment. The electrolyte leakage studies indicated that the reduced germination performance of these seeds was not due to plasmalemma disorganisation. These deleterious effects, however, were counter-balanced as seeds treated at 55, 57 and 60 QC for durations up to 60, 30 and 10 min, respectively, produced plants of superior quality than those of the control, which is ascribed to the reduction of systemically transmitted pathogens. The efficacy of the hot water treatment in reducing the levels of seed infection and improving seedling quality was enhanced by subsequent re-dehydration. The reduction in seed-associated mycoflora was maintained following storage for 1 month at both 4 QC (33% RH) and 25 QC (91% RH). However, both seed and seedling quality were adversely affected following storage even under cold, dry conditions, which may be a consequence of the pre-treatment history of the seeds, which had been cold-stored for two years prior to the experiments. Applied as a pre-sowing treatment, therefore, hot water treatment shows promise for producing a crop of superior quality, less prone to fusarial pathogenesis. This treatment may be of particular importance to Third-World subsistence communities. / Thesis (M.Sc.)-University of Natal, 1995.

A review of the literature revealed that there is a lack of depth in our knowledge of gymnospermous seeds with regard to the development and germination processes. The phytohormones, particularly the cytokinins have been implicated in these processes. The seeds of Podocarpus henkelii were thus selected as experimental material for studying the biochemical and cytokinin-like changes associated with development and germination. The development of these seeds was also followed at the ultrastructural level. These studies revealed that cellular detail within the female gametophyte only began .to form in December (early summer), approximately six weeks after fertilization had taken place. At this time some reserve protein was evident and the embryo sporophyte consisted of only a few pro-suspensor and pro-embryo cells. Concurrently, the cytokinin levels were fairly high in the female gametophyte but low in the epimatium. In both seed components two cytokinin- like compounds predominated which co-chromatographed with the free base cytokinin zeatin and its ribonucleoside. The second sample was taken in late January (mid-summer) by which time the embryo sporophyte had developed rapidly into a readily distinguishable seed component. The cellular detail indicated that much cell division had recently taken place and that the cells were currently increasing in size and accumulating starch and lipid. In the female gametophyte the soluble sugars were at the maximum level recorded during these experiments and the level of starch was increasing. The extractable cytokinin content of the seed was high at this time, particularly in the embryo sporophyte. In all three seed components cytokinin-like compounds which co-chromatographed with zeatin and ribosylzeatin were present. These high levels of cytokinin coincided with the rapid increase in both fresh and dry mass of the embryo sporophyte and female gametophyte. Ultrastructural studies of the third sample collected in mid-March (early autumn) showed that cellular changes were associated mainly with increases in cell size and the accumulation of food reserves, particularly starch. The cytokinin levels had decreased in all three seed components at this time. There was an increase in the cytokinin which co-chromatographed with glucosylzeatin in the female gametophyte. The seeds matured in late April (autumn) and had the unusual features of not drying out during maturation. Fresh seeds collected from the ground had a moisture content of ca. 62 per cent. The main food reserve was starch with relatively small amounts of protein and lipid also present. The seeds of Podocarpus henkelii germinated readily after scarification in the absence of water provided that their moisture content remained ca. 60 per cent. Seeds in which the moisture content had fallen below ca. 54 per cent required additional water for germination. The moisture content of the seeds fell rapidly under natural conditions and viability was lost below a moisture content of ca. 34 per cent. Unscarified seeds of 52 per cent moisture content placed under moist conditions at a constant 25°C took 23 weeks to achieve 68 per cent germination. These experiments showed that although the epimatium limited water uptake by the seeds it did not prevent moisture loss to the atmosphere. This appears to be the main factor contributing to the seed's inefficiency as a propagule. A small degree of after-ripening was recorded with the embryo sporophyte increasing in size with storage. This appeared to contribute to the increased rate of germination of the scarified seeds. An interesting feature of the seeds of Podocarpus henkelii is that they have the ability to fix atmospheric carbon, which is subsequently translocated from the epimatium to the female gametophyte and embryo sporophyte. The mature seeds were stored at 4°C for six weeks during which time little change had occurred at the ultrastructural level. Protein vacuoles in the embryo sporophyte had disappeared and in all three seed components cytokinin levels were low. Three days after scarification and the start of incubation, little change in cellular detail was apparent as limited rehydration was necessary due to the high moisture content. The cytokinin levels in the embryo sporophyte and epimatium had increased, whilst the levels in the female gametophyte had decreased at this time. In the embryo sporophyte lipid mobilization had commenced with these reserves apparently being metabolized within vacuoles. The rate of respiration measured in terms of increases in CO[2] evolution, increased 60 hours after the start of the incubation period, just 12 hours before ten per cent germination was recorded. Germination was accompanied by a large increase in the levels of cytokinins in the female gametophyte and embryo sporophyte. The cytokinins detected co-chromatographed with the free base cytokinin zeatin and its riboside, ribosylzeatin. Concurrently, marked ultrastructural changes were recorded with increases in the amounts of dictyosomes, endoplasmic reticulum and the formation of polyribosomes, all of which are indicative of increased metabolic activity. Similar increases in the female gametophyte were ofa lower order and occurred only after nine days of incubation. By this time the levels of cytokinins had decreased considerably. After 12 days of incubation 65 per cent of the seeds had germinated. As much of the food reserves in the female gametophyte and embryo sporophyte remained, it is suggested that these reserves are utilized for subsequent seedling establishment rather than for germination. The actual role that cytokinins play in the development and germination of these seeds is not clear. High levels of this phytohormone coincide with periods of food deposition and mobilization suggesting that they play an important part in these processes. The results of the biochemical, cytokinin and ultrastructural studies are discussed in relation to the developmental and germination processes and are compared to the data of other seeds. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1981.