Masters Degrees (Botany)

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Aspects of the influence of temperature on the desiccation responses of seeds of Zizania palustris (Wild rice)
(1996) Ntuli, Tobias M.; Berjak, Patricia.; Pammenter, Norman William.
Seeds of wild rice (Zizania palustris var. interior) have been reported to show highest survival when dehydrated at 25 QC. It has also been reported that axis cells sustained least damage at this drying temperature. In the present study, a linear relationship between drying rate and dehydration temperature was established. Whereas highest positive tetrazolium staining and lowest leakage were recorded for seeds that were dehydrated at 25 QC, maximum germination was recorded for seeds dried at 20 QC. A proportion of seeds showed the presence of glasses, irrespective of the dehydration temperatures used. Parameters of the glass to liquid transition, however, correlated with neither water content nor sugar profiles. The ratio of raffinose to sucrose was similar among all the treatments. A hydroperoxide test revealed a linear relationship between peroxide levels and temperature of drying although the levels of fatty acids were not correlated with hydroperoxide levels. Butanal levels and total aldehydes evolved, on the other hand, showed a high negative correlation with peroxide levels. Electron microscopy showed that the variability and relative abundance of peripheral membrane complexes (PMCs) was the highest for cells of embryonic axes dehydrated at 25 QC and the lowest for embryonic axes of seeds dried at 10 QC. Furthermore, intramembrane particles (IMPs) were evenly distributed in cells of axes dried at 25 or 37 QC. In contrast, membranes of cells of axes dehydrated at 10 QC showed large IMP-free areas. The relative abundance of IMPs was the highest for cells of embryonic axes dried at 25 QC, and the lowest for cells of axes dehydrated at 10 QC. From these observations, it is suggested that membrane phase transition, with the concomitant elimination of proteins, accompanies dehydration of Z. paluslris seeds at 10 QC, whereas at 37 QC peroxidation may predominate.
Development of micropropagation protocols for selected indigenous plant species..
(1995) Hannweg, Karin Fiona.; Watt, Maria Paula Mousaco Deoliveira.; Berjak, Patricia.
The herbal medicine trade is thriving in KwaZulu Natal with an ever-increasing number of people harvesting and trading in indigenous plants, especially those species with medicinal and/or magical properties. The number of plants harvested has increased whereas the size of the plants collected has decreased, resulting in low recruitment into wild populations. As a result of these two factors, species diversity has decreased. To this end, the aim of these investigations was to establish micropropagation protocols for the selected species i.e. Bowiea volubilis, Haworthia_ limifolia and Cryptocarya latifolia. In addition, hardening-off protocols were also developed. The bulbous plant, Bowiea volubilis, was propagated via organogenesis using the inflorescence stem. Bulblet formation occurred directly without an intervening callus phase. Bulblets were produced on explants on Linsmaier and Skoog (1965) (LS) medium containing 30 g.r' sucrose and either I mg.r' BAP and I mg.r' 2,4-D or 1 mg.r' BAP and 1 mg.r' NAA. Shoots and roots were induced upon transfer to the basal medium devoid of plant growth regulators. Regenerated plantlets were successfully hardened-off. Haworthia limifolia, a succulent, was propagated via direct somatic embryogenesis using leaf material. Embryo formation was induced on a modified Murashige and Skoog (1962) (MS) medium containing 20 g.r' sucrose and 1 - 5 mg.r' 2,4-D. secondary embryogenesis occurred when the explants were transferred to the basal medium supplemented with activated charcoal and devoid of growth hormones. Healthy plantlets, produced from secondary embryos, were transferred to pots and acclimatised to greenhouse conditions. A large proportion of the plantlets regenerated were vitrified and as a result, this problem was addressed by changing the medium composition or culture environment. Silica gel, when placed in the culture vessel, was the best treatment for reversal of the vitrified condition. The establishment of leaf and nodal segment cultures of Cryptocarya latifolia required extensive investigation of sterilants to reduce fungal contamination. Several fungicides were tested and a successful sterilisation protocol was established. A number of media were tested for the induction of dormant axillary buds and multiplication of shoots. The best medium for both bud induction and proliferation was MS medium containing 30 g.r1 sucrose and 1 mg.r1 BAP and 0.01 mg.r1 NAA. Callus cultures were established on MS medium containing 30 g.r1 sucrose and 3 mg.rl 2,4-D. These calli, however, were non-embryogenic. Application of the established protocols and future research strategies are discussed.
Differing responses of embryonic axes of four recalcitrant-seeded species from temperate and tropical provenances to the procedures involved in cryopreservation.
(2016) Mshengu, Thembela Mathews.; Pammenter, Norman William.; Berjak, Patricia.
Abstract available in PDF file.
The effect of provenance on the response of the recalcitrant seeds of Trichilia dregeana to drying and chilling.
(2011) Oyerinde, Rebecca Opeyemi.; Berjak, Patricia.; Pammenter, Norman William.
One of the factors that affect the post-harvest behavior of seeds is provenance. Different geographical locations are characterised by different environmental conditions, hence, plant materials harvested from different locations may respond differently to laboratoryinduced stresses. The aim of the present study was to assess the role that provenance plays on the degree of recalcitrance of a single species – a factor that needs to be taken into consideration when choices are to be made for plant germplasm conservation. In this study, seeds of Trichilia dregeana, which have been shown to display recalcitrant post-harvest behavior, were harvested from four locations that were slightly different in climatic conditions: Mtunzini (MTZN), Durban (DBN), Pietermaritzburg (PMB) and Port Edward (P.ED), all within the KwaZulu-Natal province of South Africa. Clean seeds, whose aril and seed coat have been removed, were subjected to different drying and chilling stresses and their responses to the stresses are examined. Excised embryonic axes of the seeds were also subjected to flash (very rapid) drying and their responses to the stress are shown. Although the vegetation of these locations can be categorized as being sub-tropical, the present study suggests that there may be ‘degrees’ in the sub-tropical nature based on the rainfall and maximum temperature data. The ‘degree of sub-tropicality’ is suggested to be in the order MTZN > DBN > P.ED > PMB. Mean seed size assessed as seed length, width and fresh mass varied significantly across the four regions, with seeds from MTZN being the smallest and those from P.ED being the largest. The shedding water content of the embryonic axes of the seeds for 150 min. However, shoot production, root and shoot length and dry mass accumulation showed that axes of seeds from MTZN were the most adversely affected by rapid drying while those from PMB were the least sensitive. When clean whole seeds were dried slowly by burying in silica gel for 36 h, all seeds of the four provenances still retained more than 50% of their shedding water. MTZN seeds lost viability completely from the 24 h of slow drying while seeds from the other three locations retained some viability after 36 h. The order of survival of slow drying was MTZN < DBN < P.ED = PMB. Storage of cleaned whole seeds at chilling temperatures (3oC, 6oC and 16oC) caused increase in the water content of the embryonic axes of the seeds across the four locations. All seeds of the four provenances had lost more than 50% survival after they had been stored at 3oC for 10 weeks. Survival was completely lost from the 12th week for MTZN seeds, from the 14th week for DBN and P.ED seeds and from the 16th week for PMB seeds. There was no survival recorded for seeds across the four regions at the 18th week. The seeds were able to tolerate storage at 6oC for a little longer than those stored at 3oC. Only MTZN seeds completely lost viability at the 18th week; seeds from the other three locations retained some viability throughout the 18 weeks of storage. Storage at 16oC was the most tolerable, as all seeds from the four provenances maintained some viability until the 18th week in storage, with MTZN having the least survival and those from P.ED having the highest survival. Phylogenetic analysis of the internal transcribed spacer (ITS) region of the DBN and PMB samples showed intraspecific levels of genetic variation, and were separated by a genetic distance of 0.9%, an indicator that differences in storage behavior, chilling and desiccation sensitivity between seeds obtained from different collection localities may be genetically based. This study showed that MTZN seeds were the most sensitive, while seeds from PMB and P.ED were the most tolerant, to drying and chilling treatments, respectively. The prevailing climatic conditions may have a bearing on the responses observed in this study.
Effects of some of the procedural steps of cyropreservation on cryo-recalcitrant zygotic embryos of three amaryllid species producing desiccation-sensitive seeds.
(2013) Ngobese, Nomali Ziphorah.; Berjak, Patricia.; Pammenter, Norman William.
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.
On the vacuolar system in maize roots.
(1979) Lamb, Jennifer Margaret.; Berjak, Patricia.
Root-cap cells of Zea mays L proliferate by division in the cap meristem, and subsequently differentiate and mature as they move towards the periphery of the cap, where ' they undergo autolysis and are sloughed. Vacuolar ontogeny has been shown to be complex, several different mechanisms operating not only within the root cap tissue, but within the single cells. Vacuolar initials (provacuoles) are formed in the meristem by the pinching off of single- or doublemembrane bound vesiculations of the E.R. In some instances large vacuoles appear to be formed in the mature region of the cap through the sequestering of large organelle-free regions of cytoplasm by vesicles and small cisternae, thought to be of E.R. origin. Further development of provacuoles comprises their expansion and extensive fusion, this process culminating in the formation, in a mature cell, of just one large vacuole. The vacuoles of the mature region are autophagically active, engulfing all types of cytoplasmic organelle which are subsequently lysed; these vacuoles show a positive cytochemical reaction for acid-phosphatase, further indicating that they are lysosomal in nature. The dictyosomes of the late mature cells are hypersecretory and autoradiographic and cytochemical evidence indicates that the vesicles contain an accumulation of polysaccharide. These vesicles appear to follow two secretory pathways; firstly fusion with the plasmalemma, with secretion of their content into the extra-protoplasmic space where it accumulates, finally penetrating the cell wall and middle-lamella and forming viscous polysaccharide slime on the exterior of the cap. Secondly, these vesicles appear to be engulfed by and broken down within the vacuoles. At this stage the vacuole expands considerably, and it has been postulated (Berjak and Villiers, pers. corom.) that hydrolysis of the dictyosomally-derived polysaccharide within the vacuole to monosaccharide units results in osmotic. changes leading to an influx of water into the vacuole, and its consequent expansion. Autoradiographic, cytochemical and chromatographic evidence is not inconsistent with an accumulation of monosaccharide units being at least partially responsible for the osmotic uptake of water into the swelling vacuole. Finally, the vacuolar membrane becomes discontinuous, allowing hydrolytic enzymes p~esumably contained within the vacuole to come into contact with the cytoplasm, which consequently undergoes autolysis. At this stage the cell is sloughed from the cap.
Optimising methods for embryonic axis fixation and micropropagation of Syzygium cordatum Hochst.
(2009) Premsagar, Varsha.; Berjak, Patricia.; Pammenter, Norman William.; Wesley-Smith, James.
Syzygium cordatum Hochst. (family - Myrtaceae), commonly called the umDoni (Zulu) tree, is found throughout South Africa. The tree is utilised for its fruit, bark and wood by many villagers, and this demand has placed potential pressure on existing populations. It is necessary to conserve this widely used tree before it becomes threatened by over-utilisation. Seeds of S. cordatum are recalcitrant and storable only in the short-term at 16oC over moist paper towel (hydrated storage). The study was initiated to follow deterioration of the embryonic axes, in relation to dehydrated versus and hydrated storage. However, for electron microscopic investigations, it was crucial that material was properly fixed to obtain samples that accurately represented the in vivo conditions. This proved to be challenging, as explained below, and changed the original aim of the project. The high phenolic content of S. cordatum seeds and axes makes fixation, using an aldehyde-based fixative, such as glutaraldehyde, difficult, as the aldehyde groups bind to phenolic compounds, forming large oligomers that tear out during sectioning. This causes sections to become fragmented, making viewing with the transmission electron microscope (TEM) impossible. The quest to visualise the ultrastructure, consequently became an additional focus of the project. Substituting glutaraldehyde with alternate primary fixatives including potassium permanganate (KMnO4) and 1% osmium tetroxide (OsO4) did not improve the situation. Cryo-fixation followed by freeze substitution was then attempted. Three substitution media, comprising glutaraldehyde, tannic acid, osmium tetroxide and acetone were used, all providing similar, unsatisfactory results showing ice crystal damage. Eventually, glutaraldehyde fixation was modified where samples were fixed in glutaraldehyde while being exposed to microwave energy. Results from this method of fixation were far better, with fine structure adequately preserved. A second facet of the project was aimed at producing explants alternative to seed-derived zygotic axes. Cotyledonary explants used in an attempt to produce somatic embryos, were cultured onto media which incorporated various concentrations of 2,4-D, BAP and NAA. The callus produced was sub-cultured onto regeneration media, which included NAA and BAP or PGR-free media, did not develop further. Zygotic axes cultured onto shoot multiplication medium containing BAP and NAA produced adventitious shoots which produced roots when sub-cultured onto media containing GA3.
Oxidative status and stress associated with cryopreservation of germplasm of recalcitrant-seeded species.
(2012) Naidoo, Cassandra Dasanah.; Berjak, Patricia.; Varghese, Boby.; Pammenter, Norman William.
Genetic diversity of cultivated species and their wild relatives, as well as of wild species encompasses plant genetic resources or germplasm, the ex situ preservation of which embodies a critical aspect of biological conservation. While seed storage affords an efficient ex situ conservation method, recalcitrant seeds are intolerant of desiccation and cannot be stored conventionally in seed banks. Seeds of the three indigenous tree species investigated in this study, viz. Trichilia emetica, T. dregeana and Protorhus longifolia are recalcitrant, with the species considered to be endangered. Cryopreservation, which involves storage at ultra-low temperatures of selected tissue(s) from which plants are subsequently able to be generated, is currently the only method available for long-term ex situ conservation of recalcitrant-seeded species and affords significant potential for the future. Many protocols that have been applied for the cryopreservation of the germplasm of recalcitrant zygotic embryonic axes excised from seeds of tropical/sub-tropical species have resulted in survival post-cryo which has been recorded only as root development or callus formation, with shoot formation seldom occurring. Successful cryostorage of genetic resources cannot be achieved until post-cryopreservation recovery facilitates normal seedling development, i.e. the formation of both a fully functional root and a shoot. Cryopreservation requires the utilisation of the smallest explant possible (greatest surface area to volume ratio), the most suitable for recalcitrant seeds in general being the zygotic embryonic axis. Based on preliminary studies it was demonstrated that shoot production by axes is inhibited in association with a burst of reactive oxygen species (ROS), produced in response to wounding upon excision of the axis from the cotyledons, when these are attached close to the shoot apical meristem. It was postulated that a combination of the oxidative burst at the site of excision coupled with inadequate antioxidant machinery within the recalcitrant axis tissue, precludes shoot production. It was further considered highly probable that each subsequent stressful manipulation throughout the cryopreservation process would be accompanied by a surge of uncontrolled oxidative activity within the tissue, in response to the stress. Therefore, the primary aim of the study was to investigate the underlying causes of failure of shoot production after procedures associated with cryopreservation and to focus on ways to ameliorate the consequences of unbalanced oxidative metabolism. Additionally, studies were carried out to optimise each step of the cryopreservation procedure, viz. cryoprotection, dehydration, rehydration and cooling, and subsequent recovery, in conjunction with assessment of oxidative responses, ultimately to achieve successful cryopreservation of the embryonic axes of these species. The experimental work conducted to achieve this aim assessed changes in various biomarkers of injury, those selected for this study being three ROS, viz. superoxide, the hydroxyl radical and hydrogen peroxide, after axes were exposed to various pre-treatments, cryopreservation and recovery. Concomitantly, the elicited responses of endogenous antioxidant systems accompanying these steps were assessed. Changes in the levels of ROS and antioxidant activity were determined using various biochemical assays, and these parametres, together with assessment of shoot development, were investigated after each step of the cryopreservation process. The effect of stress on oxidative metabolism was tested after exposure to pre-treatments with and without the provision of various antioxidants, viz. DMSO, ascorbic acid and cathodic water, so as to determine the efficacy of selected ROS scavengers and, in general, to develop the best protocol for cryopreservation of embryonic axes of the three species. Significant results, in terms of shoot development and regulated ROS generation, were obtained after three major processes of the cryopreservation procedure. The production of roots and shoots by excised axes of T. emetica, T. dregeana and P. longifolia after excision (75%, 80% and 75%, respectively), and by 40% of excised axes of T. dregeana after each of the two further stages, cryoprotection and desiccation, were major achievements towards cryopreservation of the recalcitrant germplasm. The modulation of ROS by ascorbic acid and cathodic protection significantly improved survival of axes of both Trichilia species. In its entirety, the present study made significant advancements towards cryopreservation of recalcitrant germplasm and also towards understanding oxidative events associated with cryogenic processing and exposure to cryogenic conditions. This study concludes that unregulated metabolism is one of the underlying causes of failure of recalcitrant germplasm represented by zygotic axes, to survive cryopreservation. The application of antioxidants and cathodic protection during cryopreservation facilitated survival that has been previously unattainable. The outcomes of this study provide an informative platform for further optimising cryopreservation procedures for the germplasm of the species investigated, and extending the work to other recalcitrant-seeded species, especially those of tropical/sub-tropical provenances.
The potential of hot water treatments for curtailing seed-associated mycoflora.
(1995) Erdey, Deon Philip.; Berjak, Patricia.; Mycock, D. S.
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.
Some aspects of biological control of seed storage fungi.
(1995) Calistru, Claudia.; Berjak, Patricia.
Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising.
Some aspects of megagametophyte development and post-shedding seed behaviour of Encephalartos natalensis (Zamiaceae)
(2009) Woodenberg, Wynston R.; Pammenter, Norman William.; Berjak, Patricia.
Very little is known about the post-shedding seed behaviour and megagametophyte development of the cycads, the most primitive extant seed-bearing plants, which pre-date the dinosaurs. In the present investigation, seeds of Encephalartos natalensis Dyer and Verdoorn were shed with relatively high mean embryo (3.33 g g-1) and megagametophyte (1.25 } 0.16 g g-1) WCs, when the developing embryo consisted primarily of the coiled, elongated suspensor bearing a rudimentary sporophyte at its tip. It was not surprising that these seeds were revealed as desiccation sensitive in the present investigation, as the embryos continued to develop after seed-shed, reaching a germinable size (.15 mm) only 4 . 6 months after seed abscission from the strobilus. Maintenance of the seeds in hydrated storage conditions was precluded by the proliferation of fungi, despite the application of the fungicide: BenlateR. Some seeds were also found to germinate in hydrated storage, despite the hard physical barrier to germination imposed by the enclosing sclerotesta. Seeds dusted with BenlateR and placed in eopen f storage in loosely closed paper bags had a longer life-span than those placed in hydrated storage; however, seeds stored in open storage were also overcome by fungi, but only around 18 months after seed-shed. Therefore, while the vigour and viability of the seeds appeared to decline slowly in the months after the embryos reached a germinable size, the life-span of stored E. natalensis seeds devoid of fungi is yet to be determined and will be the subject of further research. The current investigation also combined ultrastructural and viability retention studies to observe the post-shedding behaviour of the storage tissue, the megagametophyte. The cells of the megagametophyte became progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in an homogeneous matrix. When the development of the megagametophyte cells was analysed ultrastructurally, it was found that the unusual matrix was present from the inception of megagametophyte cellularisation, and contained microtubules and numerous very faintly-visible vesicles. Newly-formed megagametophyte cells were thus not highly vacuolated as previously thought, but dominated by an homogeneous matrix. Enzyme-gold localisation was employed in an attempt to determine the organelles responsible for the deposition of cell wall components during cellularisation of the megagametophyte. It appeared that ER-derived vesicles (and not Golgi-derived vesicles) were the principal contributors of the primary cell wall components, pectin and xylan. While cellularisation took place over approximately 1 - 2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months -when the seeds were shed whether pollination/fertilisation had recently occurred, or not. At seed-shed, the cells of the megagametophyte were nucleated and contained a few mitochondria of a metabolically-active appearance. The occurrence of aerobic metabolism in these cells was confirmed by the tetrazolium (TTZ) test. Judging from the TTZ reactivity, the viability of the megagametophyte cells of fertilised seed appeared to decline slowly in the months after seed-shed, in parallel with extension growth of the embryo. The cell layer comprising the external surface of the megagametophyte showed marked ultrastructural differences from the inner cells, and may emerge as having an ‘aleurone-like’ function. It is, however, possible that the cells of the body of the gametophyte participate actively – at least in the earlier stages of post-shedding seed development – in mobilisation of stored reserves, which must support the development of the embryonic sporophyte.
Some investigations of the responses of Quercus robur and Ekebergia capensis embryonic axes to dehydration and cryopreservation.
(2000) Walker, Marieanne Julie.; Berjak, Patricia.; Watt, Maria Paula Mousaco Deoliveira.
Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important.
Some investigations towards the cryopreservation of sugarcane germplasm.
(2009) Jaimangal, Ashika.; Pammenter, Norman William.; Berjak, Patricia.
Sugarcane has become an increasingly important crop in recent years, with South Africa featuring as one of the prominent producers. This has led to a significant growth in the South African sugarcane industry, translating into an increased demand for planting material. Although this demand is now satisfied by recent biotechnological advancements such as protocols for somatic embryogenesis to increase the production of planting material, such techniques are limited as a result of the progressive loss of the embryogenic potential of calli over time. In order to facilitate management of this material, it is desirable to develop a protocol for the long-term storage of the germplasm. This study reports on investigations of the different parameters that influenced the cryoprocess in attempts to develop a protocol for the successful cryopreservation of sugarcane somatic embryos of the 88H0019 variety. Experiments were carried out to determine in vitro culture conditions for successful induction of somatic embryos via both the direct and indirect routes of micropropagation. A suitable regeneration medium for plantlet establishment pre- and post-cooling was established (Chapter 2). Investigations were also carried out to ascertain the responses of somatic embryos to both rapid and slow dehydration techniques (Chapter 3). Finally, several cooling techniques (both slow and rapid), were applied, on partially dehydrated somatic embryos, either without, or after cryoprotection, in an attempt to achieve survival after cryopreservation of the somatic embryos (Chapter 4). Both directly- and indirectly-derived somatic embryos were converted, most successfully, on full strength Murashige and Skoog medium without addition of plant growth regulators. The initial mean water contents of directly- and indirectly-derived somatic embryos were not significantly different from each other (8.38±0.19 g g-1 and 8.45±0.33 g g-1 [dry mass basis], respectively). The percentage conversion at these water contents was also not significantly different; 97% for directly- and 98% for indirectlyinduced embryos. Slow dehydration by culture on a series of media with increasing concentrations of sucrose (from 0.2 M to 1.2 M) for a period of 48 h each was the most effective technique, with water content being reduced to 0.94±0.03 g g-1 and 0.95±0.02 g g-1 after dehydration on media containing 1.0 M sucrose, while maintaining between 98% and 100% conversion, respectively. Of the various cryoprotectants tested, proline and casamino acid had the least adverse effects on the somatic embryos. The encapsulation-vitrification cooling technique was the most efficient of all techniques employed. The best conditions involved encapsulation of embryo clumps in a solution of MS medium with 3% (w/v) Na-alginate and loading solution containing 2 M glycerol plus 0.4 M sucrose, followed by infiltration and dehydration at 0°C for various time intervals (0, 5, 10, 15, 20, 25, 30 min) with 1 ml PVS2 solution and thereafter, rapid immersion in liquid nitrogen. Under such conditions, 30% of the cryopreserved somatic embryos retained viability, going on to form callus from which shoots and roots were produced. Although somatic embryos of sugarcane of the local variety 88H0019 have proved to be recalcitrant to cryopreservation, the results obtained with explants that had been processed by encapsulation-vitrification suggest that this approach may be worth pursuing and refining.
Some invetsigations on the responses to desiccation and exposure to cryogenic temperatures of embryonic axes of Landolphia kirkii.
(2011) Kistnasamy, Provain.; Pammenter, Norman William.; Berjak, Patricia.
Landolphia kirkii is scrambling shrub forming an integral part of the flora along the coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as food source, by monkeys and rural communities and the highly recalcitrant nature of their seeds threatens the continuation of the species. In addition, the ability of the plants to produce high quality rubber makes its long-term conservation highly desirable. Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no survival had been recorded at cryogenic temperatures of below -140ºC. The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates, thawing and imbibition treatments on survival of embryonic axes excised with cotyledons completely removed, as well as with 3 mm portion of each cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was assessed by the ability for both root and shoot development in in vitro culture, the tetrazolium test and electrolyte leakage readings. At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry mass basis). All axes (with and without attached cotyledonary segments) withstood rapid (flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology regardless of the thawing temperature employed. Furthermore, analysis of the lipid composition within embryos of L. kirkii revealed negligible amounts of capric and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic temperatures. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent thawing and rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on moistened filter paper or with rehydration in water) was associated with highest survival post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75% respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to - 70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below -180ºC. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.
A study of some chilling responses of recalcitrant seeds of Avicennia marina (Forssk.) Vierh. and Ekebergia capensis Sparrm.
(2002) Lewis, Elisabeth Jacqueline.; Berjak, Patricia.; Pammenter, Norman William.
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.
The effect of developmental status and excision injury on the success of cryopreservation of germplasm from non-orthodox seeds.
(2007) Goveia, Meagan Jayne Theresa.; Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman William.
The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated.
Towards developing effective decontamination procedures for in vitro culture of embryonic axes excised from recalcitrant seeds.
(2013) Cherian, Jency.; Berjak, Patricia.; Pammenter, Norman William.
Control of seed-associated micro-organisms is vital in reducing losses of plants of economic importance. Recalcitrant seeds being metabolically active and able to be stored only under conditions of high relative humidity makes it more difficult to control contaminants. Nevertheless, means need to be developed to eliminate, or at least curtail, seed-associated fungi and bacteria. The use of biological control is a highly recommended alternative to chemical control for reducing the risk of killing beneficial organisms, as well as in terms of health and environmental hazards. Furthermore, when working with seed-derived tissues, it is extremely important to optimise a method or methods to control contamination without compromising the viability or further development of the explants. The original aim of the present study was to determine whether the biocontrol agents, EcoT® and Eco77® (commercial products of the spores of Trichoderma harzianum) would effectively control/eliminate micro-organisms from the embryonic axes of Trichilia dregeana, while promoting growth under in vitro conditions. Other means were also tested for their efficacy in controlling contaminants; these were application of Benlate®, Nipastat® (a mixture of parabens), anodic water (the anodic fraction of an electrolysed dilute solution of calcium and magnesium chloride), sodium dichloro-isocyanurate (Medi-Chlor®[NaDCC]) and alginate gel encapsulation of the embryonic axes. Prior to the experiments, fungal contaminants from the embryonic axes were isolated on potato dextrose medium and identified using light microscopy. EcoT and Eco77 were initially individually tested by co-culture as conidial suspensions with the embryonic axes. A further approach used liquid culture (potato dextrose broth) as well as solid culture medium (based on sugarcane bagasse) in/on which the strains of Trichoderma harzianum had been grown. This was aimed at testing for the possible presence of compounds released by T. harzianum into the media, which might prove to be effective in curtailing/eliminating the axis-associated microflora. Among the different treatments tested, the best method was utilised to decontaminate the embryonic axes prior to minimal-growth storage (hydrated axes encapsulated in alginate gel ‘beads’). Penicillium spp. were predominant among the different fungi isolated, which included Fusarium spp., Rhizopus spp., Aspergillus niger and Aspergillus flavus. Co-culturing with T. harzianum for 24 h was successful in terms of the survival of the embryonic axes, although the roots produced were shorter than when axes were cultured alone, but had no effect in eliminating the contaminants. Longer periods of co-culturing with T. harzianum affected the germination of the embryonic axes of T. dregeana compared with axes germinated in the absence of the biocontrol agent (control). The culture filtrate negatively affected germination of the T. dregeana embryonic axes, although it was effective against the associated contaminants. Nipastat was effective in reducing the contamination, and, depending on the concentration, did not affect germination adversely. Medi-Chlor was highly effective in eliminating all the contaminants from axes in vitro. Both these treatments were therefore used to decontaminate axes before minimal-growth storage. All the NaDCC-treated, encapsulated axes examined after 14 d hydrated storage [in Magenta boxes] and after 14-42 d in polythene bags survived; however the axes stored in aluminium foil-lined bags and Eppendorf tubes soon lost viability. The recommendation is therefore made that the decontamination treatment based on use of NaDCC (or other preparations of sodium dichloro-isocyanurate) be tested on embryonic axes of a range of recalcitrant-seeded species, and, if successful, the procedure be introduced into cryopreservation protocols. The use of NaDCC has emerged as a promising method of eliminating contaminating microflora which otherwise compromise in vitro procedures, from seed-derived explants. Furthermore, containment of decontaminated encapsulated axes in sealed polythene bags offers an apparently ideal means of temporary storage and dissemination. The results should find considerable applicability when excised embryonic axes representing the germplasm of recalcitrant seeds, are cryoconserved.
Towards development of a cryopreservation protocol for germplasm of Podocarpus henkelii.
(2012) Essack, Lubaina.; Berjak, Patricia.; Pammenter, Norman William.
The trees belonging to the genus Podocarpus, of which only four species are native to South Africa, are renowned for their superior quality timber. Prior to 1880, Podocarpus henkelii, together with P. falcatus and P. latifolius, played a significant role in the development of the country as they were heavily utilised as timber trees for the building of dwellings, furniture and other necessary items. Due to this over-exploitation in the timber trade, all the Podocarpus species in South Africa have been afforded a ‘Protected’ status on the IUCN red data list of species that are either threatened or in danger of extinction. However, despite the obvious need to conserve the threatened genetic diversity of these species, few attempts (aside from in vitro micropropagation) have been made to explore ex situ Podocarpus germplasm conservation in the long-term. Consequently, the primary aim of this study was to establish a protocol for the long-term conservation of germplasm of Podocarpus henkelii Stapf ex Dallim. Jacks. The seeds of Podocarpus henkelii exhibit recalcitrant behaviour and can therefore not be stored in conventional seed banks. This has necessitated the investigation of alternative methods of germplasm conservation with a focus on cryopreservation which is presently considered the most reliable, efficient and cost-effective means of storing the genetic resources of recalcitrant-seeded species for prolonged periods. The first objective of this study was to investigate the effect of slow (two-step) and ultra-rapid cooling on the post-thaw survival of variously treated P. henkelii embryos. The results of this investigation revealed that the rate of cooling employed had a significant effect on explant viability as none of the precultured, cryoprotected embryos that were slowly cooled survived cryostorage while some of the preconditioned embryos responded to ultra-rapid cooling (i.e. 36% shoot production and 88% callus formation). For ultra-rapid cooling, it was found that flash-drying prior to cooling was a prerequisite for survival as osmotic dehydration alone did not effectively prepare the tissues for the stresses imposed during cryostorage. Furthermore, for those flash drying intervals that yielded positive results, preconditioning explants with 10% glycerol proved the most effective pre-cooling treatment. However, due to the low recovery numbers after ultra-rapid cooling, a third cryopreservation technique i.e. cryogenic vitrification, was investigated. For cooling by vitrification, data obtained from preliminary experiments showed that precultured explants needed to be initially loaded with 18% sucrose (w/v) + 14% glycerol (v/v) for 20 min and subsequently immersed in Plant Vitrification Solution 3 (PVS3) at 0°C for 10 min prior to cooling. However, relatively low success was achieved for P. henkelii embryos cooled by vitrification as the highest post-cooling survival obtained was only 20% germination, 27% shoot formation and 37% callus formation. Due to the low post-thaw survival obtained despite the rigorous manipulations employed in the development of the slow cooling, ultra-rapid cooling and vitrification protocols, it was decided that an alternative explant should be investigated for the conservation of P. henkelii germplasm. The explant of choice was adventitious buds induced to form on, and subsequently excised from, mature P. henkelii embryos. The first objective was to develop a suitable protocol for the induction of adventitious buds on P. henkelii embryos. The medium that induced in the highest percentage of embryos (85%) to form adventitious buds consisted of Douglas-fir cotyledon revised (DCR) basal medium supplemented with 30 g L-1 sucrose, 0.05 mg L-1 NAA, 0.5 mg L-1 BA and 6 g L-1 agar. This medium also resulted in the highest average number of buds formed per embryo (i.e. 35 ± 3 buds per embryo). Once the adventitious bud induction medium was developed, it was necessary to optimise the size of adventitious bud clumps to be used as explants for cryopreservation. Three bud clump sizes were investigated: ca 3, 5 and 10 buds per clump. However, none of the bud clumps survived excision from the mother-tissue despite the investigation of three different types of bud-break media. The resultant tissue mortality is suggested to have occurred because the adventitious bud clumps were excised prior to bud break and shoot development which could have exacerbated excision-related cellular and sub-cellular damage. It was therefore decided that attempts should be made to induce adventitious buds directly on P. henkelii embryos post-cooling, thereby eliminating the possibility of potentially lethal excision-related damage. The protocols that yielded the best results after ultra-rapid cooling and cooling by vitrification were used in this experiment. For ultra-rapid cooling, embryos were first cryoprotected with 5% followed by 10% glycerol for 1 h in each and subsequently flash dried for 30 min prior to immersion in nitrogen slush. For cooling by vitrification, embryos that were first precultured on 0.3 M sucrose for 1 d were loaded with 10% glycerol + 14% sucrose (LS4). The loaded explants were then immersed in ice-cold PVS3 and maintained on ice for 10 min prior to cryostorage. The effect of each pretreatment (either independently or in combination) on adventitious bud production pre-cooling was also investigated. For both protocols the various pretreatments decreased not only the capacity of the embryos to form buds but also the average number of buds formed per embryo (i.e. 7 ± 2 buds per embryo and 14 ± 2 buds per embryo were formed on treated embryos prior to ultra-rapid cooling and cooling by vitrification, respectively). Thus, it was predicted that even if the percentage of cryopreserved embryos forming buds was minimal, the number of possible plantlets that could be regenerated from adventitious buds per cryopreserved explant would compensate for the low recovery of embryos post-cooling. However, none of the embryos that were cryopreserved by either ultra-rapid cooling or by vitrification formed adventitious buds after eight weeks in culture. The very restricted success achieved in this study despite the investigation of three cryopreservation techniques and two different explants only serves to reinforce the difficulties associated with the conservation of recalcitrant germplasm. The large size and structural complexity of P. henkelii embryos, coupled with their high water content post-shedding, are just some of the characteristics to which their intractability to the manipulations involved in the development of a successful cryopreservation protocol could be attributed. For future investigations, development of adventitious buds produced on cryopreserved root segments (as opposed to entire roots), and/or use of seedling meristems as explants which might be amenable to cryopreservation are suggested as possible avenues for the long-term conservation of P. henkelii genetic diversity.

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