The morphology and structure of P. pseudoparkeae is described in detail. The alga resembles other species in the genus but is most closely related to P. parkeae being separated from it by differences in scale structure. Important taxonomic (phylogenetic) characteristics of P. pseudoparkeae include the possession of four flagella, trichocysts, a complete covering of three scale types on the cell body, and a 3-over-1 arrangement of the basal bodies. The alga was grown successfully in a number of enriched and artificial seawater media. The alga grew well in a salinity of 35%0 but it is euryhaline and tolerated salinity levels ranging from 10 - 70%[0]. The relative growth rate (k') of the alga was significantly increased by raising the light intensity from 50 to 100 or 150 μ Em ⁻² s¯¹. At higher light intensities (200 and 300 μ Em⁻² , s¯¹ ) k' was reduced, probably through photo inhibition. The alga grew well at 20 and 25°C but could not tolerate a temperature of 30°C. The growth studies indicated that optimal growth (determined by the highest relative growth rate) was achieved in PES medium at a salinity of 35%0, a light intensity of 100 - 150 μ Em⁻² s¯¹ , and a temperature of 20 - 25°C. Under these conditions the mean doubling time (G) of the cells was 26 h. Scale structure in P. pseudoparkeae remained constant in the different seawater media used and under a range of salinity, temperature and light intensity. P. pseudoparkeae could not be grown axenically and was shown to have an absolute requirement for bacteria in culture. This bacteria/algal relationship is believed to be mutualistic because the alga also promoted the growth of the bacteria. The nature of the growth promoting factors involved are not known. Cell division In P. pseudoparkeae was similar to that described for other species in the genus. The cells remained motile throughout the cell division cycle and they divided preferentially during the dark. Cultures of the alga could at best be partially synchronized under optimal growth conditions because the shortest mean doubling time obtained was 26 h; i.e. two hours longer than the 24 h period in a 16h:8h synchrony induction photoregime. of the flagellar basal bodies, dictyosomes and nucleus {in that order}. Mitosis is characterized by an open spindle. Spindle microtubules, which are derived from the rhizoplast, are absent at telophase and no phycoplast develops. Cell division is completed within 90 min. All scale types covering the alga were produced continuously by the two dictyosomes within the cell. Scale morphogenesis was shown to be a rapid process with scales being completely formed within 10,5 min. This is the time taken for a single cisterna to pass through the dictyosome (comprising 20 cisternae). Flagellar scales were stored in a scale reservoir which was always connected with the flagellar pit via a duct. These scales were released when four new flagella developed from the replicated basal bodies. A compound microtubular rootlet was always associated with the duct of the scale reservoir. Body scales moved in vesicles from the dictyosomes directly to the plasmalemrna at the base of the flagellar pit where they were released by reverse pinocytosis. The scales of P. pseudoparkeae were shown to be pectinaceous In nature being predominantly composed of polysaccharide and containing a small amount (4%) of protein. TLC separation of sugar residues in acid hydrolysates of scales showed that the latter were composed of neutral sugars galactose, arabinose, xylose, rhamnose and a trace of fructose. Galacturonic acid is also thought to be a major constituent of the scales because they were digested with pectinase. The scale polysaccharide is sulphated. Aspartic and glutamic acid were major amino acid residues detected on scale hydrolysates analysed on an automatic amino acid analyser. The polyanionic nature of the scales is thought to underly the mechanism of external self-assembly of the scaleboundary, and to contribute to the maintenance of water and salt balance in the cell. P. pseudoparkeae reproduces asexually by binary fission or by producing non-motile, thick-walled cysts. Cysts developed spontaneously in cultures so that the stimuli causing encystment are not known. In fresh medium mature cysts released four motile cells which regenerated the motile phase. The cyst wall is bilayered. The outlet layer of the cyst wall has the properties of sporopollenin while the inner layer of the cyst wall has the same staining properties as scales and is possibly of similar composition. EDX analyses of the cyst wall showed that it is Tich in calcium and sulphur. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1985.

A review of the literature pertaining to cut carnation flower senescence and the regulatory role of plant hormones in this process revealed the value of this system in physiological studies. Carnation flower senescence is a good example of correlative senescence and therefore this final development stage involves an interaction between flower parts dying at the expense of the development of others. Due to the survival value of the seed, ovary growth occurs to the detriment of the surrounding flower parts especially the petals, the flower part that determines vaselife. This senescence strategy occurs, although at a later stage, even when pollination is unsuccessful. Additional ethylene applied using 2-chloroethyl phosphonic acid, which when incorporated into plant tissue produces ethylene, accelerated carnation flower senescence. If the carnation flowers are treated with silver thiosulphate,which prevents ethylene action,and ethanol,which inhibits ethylene biosynthesis,petal longevity is extended to the detriment of ovary growth. Correlating the physical appearance of the flowers in the presence and absence of ethylene with dry mass and labelled sucrose analyses, carbohydrate movement appeared to be a major event during the senescence of this cut flower. Such a conclusion could not be reached on dry mass analyses alone as the photosynthetic organs of the carnation flower contribute to the carbohydrate pool in the first days following harvest. Furthermore the respiratory pattern of the flower is not a steady decline. Concomitant with the natural ethylene emanation as the petals irreversibly wilt, so the respiratory rate increases. On the other hand, the respiratory rate is greatly reduced with silver thiosulphate and ethanol treatment. In the presence of ethylene, together with the growth of the ovary there is an influx of carbohydrates from all the flower parts including the petals into the ovary. With silver thiosulphate and ethanol treatment the petals become the dominant carbohydrate sink. It thus appears that insufficient carbohydrates moving to the ovary may be the cause of the lack of ovary development. However , an experiment with isolated cultured ovaries on a modified MILLER'S (1965) medium lacking in plant hormones but with a range of sucrose concentrations showed that sucrose alone cannot stimulate ovary growth. The mechanism by which this source-sink relationship is determined appears to be controlled from the sink. The source organs contribute carbohydrates that are in excess of their metabolic needs. Acid invertase activity, maintaining the sucrose gradient into the sink, was considered as a mechanism by which sink strength could be controlled due to the parallel in other plant systems between the activity of this enzyme and sink strength. On investigation the levels of acid invertase activity are higher in the ovaries of senescing carnations than in the petals. This balance of invertase activity was reached mainly due to a decline in petal invertase activity. However, as silver thiosulphate treatment lowered the level of acid invertase activity in the ovary and this flower part was not the dominant sink with this treatment, acid invertase activity appears to contribute to sink activity in the senescing carnation flower. Nevertheless due to the immobility of sucrose through membranes, for the passive movement of sucrose down a concentration gradient, membrane permeability to sucrose would have to be altered. This is a possible role of the plant hormones and specific ions. Furthermore, this ovary growth was correlated with chloroplast development in the ovary wall. In the presence of ethylene 'greening' or an increase in chlorophyll content during flower senescence was measured. This increase in the chlorophyll content did not occur in the silver thiosulphate and ethanol treated carnations. Relating this to chloroplast development, an electron microscope study showed that in the presence of ethylene the original amyloplast present at harvest developed into a chloroplast with thylakoids stacked into grana. With the ethylene inhibitory treatments, although thylakoids developed in the ovary wall chloroplasts, grana did not form. As chlorophyll is synthesised in the thylakoids, this chloroplast structure correlated with the chlorophyll measurements. The results of the parameters measured during the senescence of the cut carnation flower suggested that the other plant hormones besides ethylene were involved in this process. Endogenous cytokinin measurements showed that, overall, the level within the cut flower declined as the flower senesced. The ovary cytokinin levels did not steadily decline but increased as the petals irreversibly wilted. This peak of cytokinin activity was common to ovaries of flowers treated with 2-chloroethyl phosphonic acid and naphthalene acetic acid, treatments that accelerated senescence. Previous workers showed that a silver thiosulphate treatment prevented this increase in cytokinin activity in the ovary. This, together with the lack of ovary development, suggests that the ovary cytokinin activity may be a crucial event in the regulation of carnation flower senescence. To confirm such a hypothesis zeatin was injected into the ovary but was found ineffective in mobilising sucrose and accelerating petal senescence. It was only when both zeatin and indoleacetic acid were applied to the ovary that sucrose mobilisation and accelerated petal senescence occurred. Thus auxins together with cytokinins appear important in ovary development. The importance of the presence of auxin in ovary development was further recognised by a naphthalene acetic acid treatment being far more effective in ~timulating the growth of isolated cultured ovaries than kinetin. Auxin treatment increased the size of the cells within the ovary wall and the development of the chloroplasts within these cells to a greater extent compared to control and kinetintreated ovaries. It was thus hypothesised that the auxin levels in the ovary were protected against conjugation by the presence of adequate levels of cytokinins. When the cytokinin levels dropped, as in the petals, ethylene could then accelerate auxin conjugation resulting in a retardation of growth. Sink tissues, such as the ovary, with a higher cytokinin and hence auxin content, may utilise mobilised assimilates from the petals thus contributing to petal senescence. To further prove this hypothesis an investigation into the site of ethylene action using the silver ion as a tool was initiated. A review of the histochemical and histological literature revealed that common silver binding sites in plants included sulphydryl groups, chloride ions, ascorbic acid and invertase. Each was considered as potential channels via which ethylene could effect its physiological response but no conclusion was reached. Because of this a decision on the importance of the translocatory path of a ten minute silver thiosul phate pulse within the flowerhead and its accumulation within the receptacle could not be reached. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1985.

Thesis (MSc (Botany and Zoology))--University of Stellenbosch, 2005. / The main substrate use during shoot establishment from the lateral bud of sugarcane setts and enzymes involved in sucrose metabolism were investigated in the shoots and the internodes acting as source of carbohydrates. Radiolabelling studies were conducted to investigate the metabolism of sucrose and glucose during shoot establishment. The internode’s total dry mass over the 21-day of shoot establishment period was reduced by 25% and 30% in plants incubated in dark/light and total darkness, respectively.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Oxalis L. has two centres of diversity, one in South-Central America and the other in southern Africa. Previous palynological studies of southern Africa Oxalis revealed four main pollen types, namely rugulate-reticulate, micro-rugulate-spinate, reticulate and supra-areolate (Dreyer 1996). The reticulate pollen type is further divided into 15 subtypes, out of which five have a monotypic status. The supra-areolate pollen type is divided into four subtypes based on exine structure. The reticulate pollen type is the most common pollen type within the South African members of Oxalis. The three remaining main pollen types display more complex exine structures and are therefore considered more derived than the reticulate pollen type. The present study assessed the pollen of 50 American Oxalis species with three main objectives: 1. To observe pollen type variations among American members of Oxalis, 2. To compare pollen types from the two centres of diversity, and 3. To assess which centre of diversity house the palynologically more advanced species of Oxalis. Two main pollen types are recorded from the present study, namely reticulate and verrucate pollen types. The reticulate pollen type could be further divided into 11 subtypes. Out of the 11 subtypes observed, nine also occur among South African members of Oxalis, while two types are only observed in the American members of Oxalis. The verrucate pollen type is found in a single American taxon and displays a more complex exine structure than the reticulate pollen type. In this study the reticulate pollen type proved to be the most common pollen type among the American members of Oxalis. The South African members of Oxalis display more complex pollen types than the American members of the genus. / AFRIKAANSE OPSOMMING: Oxalis L. het twee diversiteitsentrums, een in Suid-Sentraal Amerika en die ander in suidelike Afrika. Palinologiese studies van suidelike Afrika Oxalis-taksa toon vier hoofstuifmeeltipes, naamlik gerimpel-netvormig, fyn-gerimpel-stekelrig, netvormig en supra-areolêr (Dreyer 1996). Die netvormige stuifmeeltipe word verder verdeel in 15 subtipes. Vyfvan hierdie tipes het 'n monotipiese status. Die supra-areolêre stuifmeeltipe word verdeel in vier subtipes gebaseer op eksienstruktuur. Die netvormige stuifmeeltipe is die mees algemene stuifmeeltipe aanwesig in die Suid Afrikaanse Oxalis-taksa. Die drie oorblywende hoofstuifmeeltipes toon 'n meer komplekse eksienstruktuur en word as meer gevorderd as die netvormig stuilmeeltipe beskou. In die huidige studie is stuifmeelkorrels van 50 Amerikaanse Oxalis spesies bestudeer met drie doelstellings in gedagte: 1. Om die variasie in die stuifmeeltipes van die Amerikaanse spesies van Oxalis te bestudeer, 2. Om die stuifmeeltipes van die twee diversiteitsentrurns te vergelyk, en 3. Om vas te stel watter diversiteitsentrum het palinologies die meer gevorderde spesies van die genus Oxalis. In die huidige studie is twee hoofstuifmeeltipes onderskei, naamlik netvormige en verrukate stuifmeeltipes. Die netvormige stuifmeeltipe is verder verdeel in 11 subtipes. Van die 11 subtipes wat onderskei is, kom nege tipes ook in die Suid Afrikaanse taksa van Oxalis voor, terwyl twee stuifmeeltipes slegs by die Amerikaanse soorte van Oxalis voorkom. Die verrukate stuifmeeltipe is slegs in 'n enkele Amerikaanse spesie gevind. Hierdie tipe toon 'n meer komplekse eksienstruktuur as die netvormige stuifmeeltipe. In die huidige studie het die netvormige stuifmeeltipe gebleik die mees algemene stuifmeeltipe in die Amerikaanse Oxalis-spesies te wees. Die Suid Afrikaanse taksa van Oxalis toon meer komplekse stuifmeeltipes as die Amerikaanse taksa.

Thesis (DPhil)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: A biosytematic revision of the seven minor genera of the Hyacinthaceae, including twenty-two species, was undertaken. Muller-Doblies & Muller-Doblies (1997) considered these seven genera (Amphisiphon Barker, Androsiphon Schltr., Daubenya Lindl., Massonia Thunb. ex Houtt., Neobakeria SChItL, Polyxena Kunth and Whiteheadia Harv.) together with the genus Eucomis L'Herit. to form the subtribe Massoniinae of the tribe Massonieae. Previous revisions of the group were based only on morphological characters (Jessop 1976; Muller-Doblies & Muller-Doblies 1997). The subtribe Massoniinae is characterised by the large variety of floral forms exhibited by the different species in the group. In the past this has led to the establishment of many monotypic genera for species thought to have unique floral structures. Morphological, leaf anatomical, palynological, geographical and molecular data were studied in order to delimit the taxa and determine the phylogenetic relationships within the group. This showed that most of the unique floral structures are probably only adaptations to pollination strategies and all except one of the monotypic genera are now placed in the genus Daubenya. In the genus Massonia there is a great deal of variation in leaf morphology and this resulted in the establishment of many invalid species, now mostly reduced to synonymy. A new species was described, several name changes made and several species were reduced to synonymy. / AFRIKAANSE OPSOMMING: 'n Biosistematiese hersiening van die sewe kleiner genera van die Hyacinthaceae, insluitende twee-en-twintig spesies, is onderneem. Muller-Doblies & Muller-Doblies (1997) sluit hierdie sewe genera (Amphisphon Barker, Androsiphon Schltr., Daubenya Lindl., Massonia Thunb. ex Houtt., Neobakeria Schltr., Polyxena Kunth and Whiteheadia Harv.) saam met die genus Eucomis L'Herit. in die subtribus Massoniinae van die tribus Massonieae in. Vorige hersienings van die groep was meestal net op morfologiese kenmerke gebaseer (Jessop 1976; Muller-Doblies & Muller-Doblies 1997). Die subtribus Massoniinae word gekenmerk deur die groot variasie in blomstrukture wat by die verskillende spesies in die groep voorkom. In die verlede het dit gelei tot die beskrywing van verskeie monotipiese genera gegrond op wat geblyk het, unieke blomstrukture te wees. Morfologiese, blaar anatomiese, palinologiese, geografiese en molekulere data is bestudeer om die verskillende taksons af te baken en terselfdertyd die filogenetiese verwantskappe binne die groep te bepaal. Dit het aangetoon dat die unieke blomkenmerke eerder aanpassings aan bestuiwings-strategiee is en dat al hierdie monotipiese genera, behalwe een tot die genus Daubenya behoort. In die genus Massonia is daar baie variasie in blaarmorfologie en dit het veroorsaak dat 'n groot aantal spesies beskryf is, waarvan baie nou as sinonieme beskou word. Een nuwe spesies is beskryf, verskeie naarnsveranderinge is gemaak, en 'n aantal van die spesies is tot sinonieme verander.

The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

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. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.

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. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Cavendish bananas are the most important sub-group of all bananas. They includes more than 30% of the global banana production and almost all bananas exported are of the Cavendish type. This sub-group is also an important food source. Most of the fruit is consumed locally, such that only 35% enters the international market. To meet the regular demand for domestic consumption and market supply there must be a reliable production strategy. The technique of tissue culture is a better option than conventional propagation techniques. However. the high incidence of somaclonal variation among plants derived from tissue culture is a problem for commercial producers. Several factors such as genotype, tissue source, duration in culture, and the tissue culture technique employed, cause somaclonal variation. The impact of plant growth regulators on somaclonal variation was studied on Cavendish banana cv. 'Zelig' obtained from African Biotechnologies Ltd., South Africa. In vitro grown plants at the 4th multiplication cycle were supplied for the investigation. The first component of the investigation dealt with the effect of types of plant growth regulators. Combinations of the auxins IAA, IBA and NAA with the cytokinins BA and TDZ were used to culture the plants for ten multiplication cycles. Plants were then randomly selected to collect leaf material for DNA extraction and RAPD analysis. The second aim of this study was to investigate the effect of the cytokinin BA on plantlets subcultured over 5-10 multiplication cycles. The auxin IAA at the concentration of 2 mgl-1 was combined with BA at concentrations of 2.5, 5.0 and 7.5 mgl-1. Plants were analyzed at each level of subculture from the 5th to the 10th cycle for respective cytokinin concentrations. Plants were then randomly selected for the collection of leaf material for DNA extraction and RAPD analysis. DNA was extracted from leaf tissue of in vitro grown plants using a modified CTAB extraction procedure. DNA amplification products were scored for the presence and absence of bands in a particular locus. Results were clustered according to their similarities. The relationship between multiplication rate and variation was assessed using correlation analysis. Results of the investigation showed that treatments with higher multiplication rates produced higher rates of variation. A variation rate of 55% was recorded for treatments containing IAA and BA. A higher rate of variation (72%) was identified in the treatment with IAA (2mgl-1) plus BA (7.5 mgl-1) over 10 cycles. In all cases the dwarf off-type was the most common type of variant obtained, contributing 87.7% of the total amount of variation. The dwarf specific marker (OPJ-041500) reported previously in Williams Cavendish was identified in cv. 'Zelig' in this study. Another band similar in size was amplified by primer OPC-15 and named OPC-151500 . This band consistently appeared in all the normal plants and was absent in all the dwarf types and hence could be used as a dwarf specific marker. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Pteroceramium, a descriptive name given to an undescribed winged species closely related to Ceramium, has uniaxial filamentous thallus construction with pseudodichotomous branching. Alternate branches become dominant. This pattern of growth is referred to as cellulosympodial growth. All growth is from an apical cell which cuts off subapical cells. The subapical cells develop into axial cells. Each axial cell cuts off six pericentral cells in a ring around its apical pole. The pericentral cells divide further to form the cortical band. Pc1 always forms on the outer face of the thallus as determined by the preceding pseudodichotomy and gives rise to the larger outer wing which is a lateral expansion of the cortical band. The smaller inner wing forms from Pc6 on the inner face. The other pericentral cells give rise apically to uniseriate spines. The pericentral cells also give rise to rhizoids and adventitious lateral branches. Each axial cell has a large central vacuole with a few peripheral chloroplasts, mitochondria and floridean starch granules. The smaller wing cells have a much denser cytoplasm with fewer small vacuoles, many chloroplasts which are more closely packed together and more floridean starch granules than axial cells. Chloroplasts have a typical Rhodophyta ultrastructure with single, evenly spaced thylakoids with phycobilisomes. Pit connections have a plug core but no plug cap. Pteroceramium has a typical Polysiphonia-type triphasic life history. The carposporophyte is naked and tetraspores are produced in a characteristic decussate cruciate arrangement. The effect of a number of physical and chemical factors on growth and morphogenesis was investigated. Pteroceramium grew best at irradiance levels between 79 μmol m⁻² S¯¹ and 129 μmol m⁻² S¯¹ with growth being limited at 30 μmol m⁻² S-I. The largest axial cells and wings were obtained from the material grown at 79 μmol m⁻² S¯¹ and the smallest measurements for material grown at 129 μmol m⁻² S¯¹. Monochromatic light fields of red, green and blue caused reduced growth rates compared to the control replicates grown in a white light from both incandescent and fluorescent lights. Light quality had no effect on morphogenesis. The critical daylength for maximum rates of cell elongation was 10 hours or longer, although 16 hours light caused a decrease in final axial cell volume. Optimum temperatures for growth of Pteroceramium were between 20°C and 25°C with growth decreasing at 15°C and 30°C. Axial cell volume was reduced and wing size was stunted at these two extreme temperatures tested. Scouring by sand caused axial cells to decrease in volume although the wings were unaffected. Smothering by sand did not prevent growth although axial cells and wings were greatly decreased in size, with the wings consisting of only one or two other cells. Tumbling to disrupt gravity did not affect the angle of each pseudodichotomy. Decreased levels of nitrogen and phosphorus limited growth but had little effect on axial cell volume and wing development. Pteroceramium was stenohaline with maximum growth at 34°/[00] and reduced growth at 300/[00] and 40°/[00]. Pteroceramium grew best at pH 7.5 and pH 8.5 with decreased growth at pH 6.5 and pH 5.5. The various pHs tested had little effect on morphogenesis. The best photosynthetic responses were obtained from material preconditioned at 80 μmol m⁻² S¯¹ compared with that at 30 μmol m⁻² S¯¹ and 150 μmol m⁻² S¯¹. There was a decrease in pigment content with increasing irradiance at which the alga was grown. Phycoerythrin was the dominant pigment. Exposure to a high irradiance (3000 μmol m⁻² S¯¹) for 30 minutes or longer inhibited photosynthesis. Plants did not fully recover even 24 hours later, indicating that this damage was permanent. Pteroceramium was able to acclimatize slowly over a week to temperature changes within the range of 15°C to 25°C. Rapid increases of 5°C within this temperature range increased photosynthetic performance and a rapid drop of 5°C decreased photosynthetic performance. However, a 10°C increase or drop reduced Pteroceramium's photosynthetic performance. Photosynthetic rates were decreased in alkaline conditions and increased in acidic conditions. Pteroceramium has well defined developmental patterns with basal band growth of axial cells and tip growth in the rhizoids. The pericentral cells are formed in a set sequence similar to Ceramium species with Pcl forming on the outer face, Pc2 and Pc3 forming on the lower and upper surface nearest to Pel respectively, Pc4 and PcS forming on the lower and upper surface respectively farthest from Pel, and Pc6 forming on the inner face. This sequence is unaffected by the direction of illumination or gravity. Exogenous application of plant hormones (IAA, GA3 and kinetin) in the concentration range of 10[-9] M to 10[-5] M had no effect on growth and morphogenesis in Pteroceramium. Application of polyamines and their precursors caused a decrease in growth and a reduction in cell size at concentrations higher than 10[-4] M. Polyamine inhibitors caused a reduction in growth and cell size at concentrations higher than 10[-5] M. Arginine increased growth at concentrations 10[-5] M and 10[-6] M. High power liquid chromatography (HPLC) separation of Pteroceramium extracts indicated that spermidine was present in Pteroceramium at approximately 38 μg spermidine g¯¹ fresh weight. The apical tip exerts an apical dominance effect on the subordinate branches, suppressing their elongation. Removal of the dominant apical tip increased adventitious branch formation. This effect was not reversed by application of exogenous IAA at concentrations of 10[-9] M to 10[-4] M. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.