Enset (Ensete ventricosum) is an important food crop that is cultivated in Ethiopia. In vitro propagation: zygotic embryo culture, shoot tip culture, callus culture and somatic embryogenesis were investigated for this crop. Forty four percent germination of excised embryos of stored seeds of enset genotype Oniya was obtained when the embryos were placed horizontally on the medium that was supplemented with 0.5 mg l ̄¹BA and 0.2 mg lˉ¹ lAA, after germination of intact seeds could not be achieved. Over 85% embryos, excised from seeds of two wild enset genotypes shortly after seed harvest, were germinated on MS medium with and without plant growth regulators (PGRs). Addition of 5 g lˉ¹ activated charcoal (AC) prevented blackening of germinating zygotic embryos and improved in vitro growth of the seedlings. Contamination of culture was reduced to a tolerable level (below 7%) when eight to ten mm long shoot tips from greenhouse-grown suckers were decontaminated for 15 min in 3.5% sodium hypochlorite and rinsed three times with sterile distilled water. However, this contamination method was not sufficient to decontaminate shoot tips from field-grown suckers. Avoiding injury to the apical domes of the shoot tips at the initiation stage, addition of 7 g lˉ¹ AC to the medium and initiation of the shoot tips for two months before splitting for multiplication considerably decreased blackening and formation of callus for genotype Keberia and Mazia. Three to five normal shoots per shoot tip were produced when halved shoot tips from in vitro germinated seedlings of enset genotype Oniya was cultured on gelled and in liquid medium and when halved shoot tips of greenhouse-grown genotype Mazia were cultured in a liquid medium. One to two shoots/buds per shoot tip were regenerated from halved shoot tips of greenhouse-grown suckers on gelled medium for genotypes Keberia, Oniya and Mazia. The presence of BA did not result in a significant increase in the number of shoots per shoot tip both with intact and halved shoot tips. Therefore, wounding the apical dome by splitting appears necessary to release lateral buds. Both blackening of explants in the presence of AC and contamination of culture in vitro were not observed with in vitro grown plant material. Callus was produced on MS medium supplemented with 0.5 mg lˉ¹ BA + 0.2 mg lˉ¹l lAA from zygotic embryos of stored seeds of enset. Adventitious shoots from the callus were regenerated in the light on MS medium lacking PGRs. Embryogenic callus was obtained from shoot tips of genotype Mazia on MS medium with 0.5 mg lˉ¹ BA + 0.2 mg lˉ¹ lAA + 0.2 mg l ˉ¹2, 4-D. A large number of somatic embryos were produced from the embryogenic callus. The results of these studies can be used in enset clonal multiplication, conservation of germplasm and breeding of the crop. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somaclonal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency of both embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g ℓ ¯¹ sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. Bulbing of the shoots was further enhanced using higher than normal levels of sucrose i.e. 6% and 5 g ℓ ¯¹ activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of 25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg ℓ ¯¹ BA resulted in multiple shoot development. In a liquid-shake culture system, this same multiplication medium induced the formation of meristemoid clusters whose rates of proliferation were higher than that achieved for shoot multiplication on either solid or static liquid media. The advantage of using meristematic clusters is that shoot hyperhydricity is avoided. Furthermore, the clusters can be mechanically separated; making the system ideal for automated plant production. Shoot morphogenesis, followed by the formation of bulblets occurred on solid MS media containing activated charcoal or high concentrations (6%) of sucrose. The induction of bulblets by sucrose was, however, slower, which may be beneficial for long-term storage and conservation ex situ. Compared to smaller bulblets, bulblets with a diameter of approximately 9 mm, acclimatized readily and grew rapidly after transferring them to the soil in greenhouse conditions. Biotechnological processes such as cell and tissue culture provide an ideal system for producing secondary metabolites, especially where their production in situ is hampered by poor resource availability or when chemical synthesis is difficult. In vitro produced Crinum moorei bulblets were found to contain nine alkaloids of the Amaryllidaceae type; three of which were released into the culture medium. Light was essential for alkaloid biosynthesis while the inclusion of BA and activated charcoal stimulated the production of specific alkaloids. / Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2002.

Senescence was examined in the economic weed Bidens pilosa, with the objectives to a) determine the effects of deflowering and defruiting on growth, chlorophyll content, photosynthesis and transpiration; b) to identify the stage of development of the head at which the flowers, seeds/fruit produce senescence signals; and c) to test for senescence activity in plant extracts made from the receptacles and leaves of Bidens pilosa. Total chlorophyll content in the controls, in association with the development of fruit, was lower in the final harvests when compared with earlier harvests in both pot and field-grown plant experiments. Deflowered Bidens pilosa plants had a higher chlorophyll concentration than both defruited and control plants in both pot and field-grown plants. Stem death of the control plants was higher than that of deflowered plants in both field and pot experiments. The present results suggest that deflowering is essential if the leaves are to be harvested commercially because it retards senescence and maintains growth. Fruit and flower heads were responsible for the reduction in leaf and stem growth after flowering in Bidens pilosa. Removing these organs slowed plant decline, suggesting that the flower head and especially the fruit are responsible for senescence. In contrast, the fruit were the main organs responsible for the decline in leaf chlorophyll concentration. In pot-grown plants in full sunlight, photosynthesis and transpiration were low in deflowered plants compared with the control and defruited plants 45 days after treatment, and it coincided with a low stomatal conductance. These results suggest that stomatal conductance played a role in lowering photosynthesis in deflowered plants. In contrast, the control plants had a higher stomatal conductance than deflowered plants 75 days after treatment, yet photosynthesis and transpiration rates were the same in both treatments. Thus stomatal conductance alone does not successfully explain differences in photosynthesis in these treatments. The dry weight of head with mature dry fruit was higher in plants grown at high light intensities than at medium or low light intensities. It coincided with a greater decline in chlorophyll concentration in the leaf nearest to the head and fruit. In contrast, photosynthesis was the same at all light intensities in the leaf nearest to the head and fruit. This suggests that high light accelerated the process of fruit maturation of the fruit which then influenced senescence in the leaf nearest to the flower head. Ethanolic and water extracts of senescent receptacles purified using paper chromatography, induced senescence of leaves in light but not in the dark. In ethanolic extracts, activity was detected in R[f]s 0.1, 0.2 and 0.3. In water extracts, activity was detected in R[f] 0.1. Senescent leaf extracts purified using column chromatography also induced senescence in light under greenhouse conditions. At high concentrations, activity was detected in fraction 10 eluted with ethyl acetate: methanol (55:45); fraction 11 eluted with ethyl acetate: methanol (50:50); fraction 12 eluted with methanol (100%) and in fraction 13 eluted with ethylacetate : isopropanol: water: acetic acid (52:28:28:4). Under growth room conditions, activity was detected in fractions 12, eluted with methanol (100%) and 13, eluted with ethyl acetate: isopropanol: water: acetic acid (52:28:28:4) in the presence of light. Fraction 1 (R[f] 0.00-0.10) from senescent receptacles, non-senescent and senescent leaves, obtained following thin layer chromatography of ethanolic extracts induced senescence under light. Fraction 1 was eluted with methanol. This fraction lacked activity when eluted with ethyl acetate. Fraction 4 (R[f] 0.25 - 0.35) from non-senescent leaf extracts, which co-chromatographed with 4-chloroindole acetic acid, gave activity in bean cuttings kept under continuous low light. Senescent leaf extracts showed no activity. Fraction 7 (R[f] 0.9 - 1.0) from non-senescent leaf extracts, also induced senescence in bean cuttings under light. The same Fraction from senescent leaf extracts lacked activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

The initiation and development of adventitious roots in cuttings are highly complex processes, influenced by both endogenous and exogenous factors. These vary from the environmental factors prior to the striking of the cutting, to the anatomical and physiological factors, within the stem. Encompassed are the nutrient status, physiological age and degree of differentiation of the tissues, and the balance of endogenous rooting inhibitors and/or promoters (including hormones). The role of cytokinins in root initiation and development has been perceived as that of an inhibitor. This investigation considered several aspects of the role played by cytokinins in the process of root development. A qualitative/quantitative analysis of the cytokinin-like activity in stem cuttings of several plants, including both easy- and difficult-to-root species was conducted on a comparative basis. There was no clear correlation between the type / level of cytokinins detected in the cuttings and the relative ease of root formation. Both qualitative and quantitative changes in the compounds exhibiting activity in the soybean callus bioassay were observed over the period of root formation in Impatiens stem cuttings. The effects on root formation in cuttings of exogenously applied auxins and cytokinins were investigated. Auxins generally promoted root number and elongation at relatively high concentrations (10[-4] M), but showed less effect on lateral root initiation and development. At high concentrations, cytokinins strongly inhibited root development, but did promote lateral root growth. In suspension culture, the effect of these hormones differed slightly, with IAA and IBA having no significant effect on root development, but NAA strongly stimulating lateral root initiation. Zeatin (10¯¹¹ M) significantly increased root length and the number of lateral roots produced. The effect of treatment of the stem cuttings with potassium permanganate and centrifugation was examined. While both these treatments have been perceived to increase root production in cuttings, no significant improvement in rooting ability following centrifugation (relative to the control) was observed. Impatiens cuttings centrifuged in the presence of distilled water showed a significantly reduced rooting ability relative to those centrifuged in the dry state. Treatment with an 8-hour pulse in 0.05 % potassium permanganate significantly increased the average root length. These treatments had an effect on the cytokinin levels and distribution in the stem cuttings. Slightly higher levels of cytokinins were associated with the increase in root number and length in both experiments. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

The prospect of integrating transgenic eucalypts with conventional breeding programmes is of value to the Plantation Forestry and Forest Products Industries. However, significant progress in this regard has still to be reported, one constraint is the lack of appropriate high yielding regeneration culture methods for clonal material. Such was the main aim of the present study. The strategy was to develop a suitable protocol using in vitro shoots of an E. grandis x E. urophy/la clone (GU185) and thereafter to test its applicability to other clones. Explants from greenhouseestablished cuttings provided the in vitro shoots, which were multiplied via axillary bud proliferation either on semi-solid medium or using a RIT A system. To determine the best conditions for callus and shoot regeneration, parameters such as vessels (petri dishes and tubes) and types and levels of plant growth regulators were tested. The best callus production (100%) and shoot regeneration (78.9 - 100% callus with shoots) for GU185 occurred on MS, 30 g rl sucrose, 4 g rl Gelrite, 5 mg rl IAA and 0.25 mg rl BAP. Parameters tested to identify the most suitable explants for indirect organogenesis were the age of parent plants, different systems to generate in vitro shoots, elongation status of explants, 1 sI and 2nd generation in vitro shoots and the use of hyperhydric shoots. Of these, the most suitable explants for indirect organogenesis were shoots from axillary bud multiplication of 3-month-old parent plants using the semi-solid system (33 shoots/dish). Up to 90% rooting was achieved on 1f4 MS (Murashige and Skoog, 1962), 15 g rl sucrose, 0.1 mg rl biotin, 0.1 mg rl calcium pantothenate, 4 g rl Gelrite and mA. The highest rooting was obtained when regenerated shoots were first multiplied and then placed on medium without plant growth regulators for one week, before transfer to root induction medium containing 0.1 - 0.5 mg rl mA. Acclimatization success was 95% when rooted shoots were placed in pots with a rooting mix (2 perlite: 1 coir) enclosed in plastic bags and the humidity was gradually reduced over four weeks. The developed indirect organogenesis protocol appeared to have a broad general application, although the tested clones exhibited a genotype-dependent response, with GU180, GUI77 and TAG31 producing fewer shoots (9, 6 and 7 shoots/dish) than ZG14 and GU185 (24 and 18 shoots/dish). Similarly high levels of rooting were obtained for TAG3l (93.8%) and ZG14 (90%) and for hardening-off (90.7% for TAG31 and 91.4% for ZG14). / Thesis (M.Sc.)-University of KwaZulu-Natal, 2004.

Members of the genus Ledebouria Roth (Hyacinthaceae), which occur in South Africa, are revised. This genus occurs throughout Africa, India and Madagascar. 33 Species are recognized and placed into nine provisional infrageneric groups. A multidisciplinary approach was used in an attempt to provide natural groupings. The following characters were analysed; morphology, micromorphology, palynology and caryology. Aspects of ovary structure and leaf micromorphology proved especially useful in the synthesis of infrageneric and specific concepts. Keys, descriptions, illustrations, distributional, ecological, medicinal and toxicological data are provided. This study is based on plants in their natural habitat, cultivated specimens and representative herbarium specimens from herbaria in South Africa and in Europe. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1993.

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. / Thesis (M.Sc.)-University of Natal, Durban, 1996.

Ocimum obovatum E. Mey ex Benth. var. obovatum is a traditionally used medicinal plant that grows along the KwaZulu-Natal coast and the western Cape of South Africa. The plant is noted for its hair restoration properties, remedy for infantile abdominal pain and cramps and its use as an enema to treat epigastric conditions in children. The aims of this study were to document the micromorphology and ultrastructure of the foliar secretory structures responsible for the production and secretion of the essential oils and chemical composition of the secretion, which gives the plant a distinct aroma. It is believed that these oils contain the active ingredients that contribute to the medicinal properties of the plant. A variety of microscopic methods and histochemical and phytochemical tests were used to achieve this. Leaves in all stages of development were pubescent and gland dots, characteristic of plants in the genus, were found on both adaxial and abaxial surfaces. Three types of trichomes were found on both leaf surfaces across all stages of development; non-glandular trichomes and two types of glandular trichomes. Non-glandular trichomes are single, multicellular and uniseriate with microornamentation and a supportive cellular pedestal. The glandular trichomes consisted of peltate and capitate trichomes. Peltate trichomes are made up of four head cells and a very small basal cell that gives the glands the appearance of being sessile. The capitate trichomes were further divided into two types based on the morphology of the glands. Type I capitate trichomes are smaller than the larger peltate trichomes and are composed of one basal cell and a head consisting of two broad head cells. Type II capitate trichomes consisted of one basal cell, one stalk cell and a single oval head cell. Histochemical tests showed that peltate and Type I capitate trichomes have cutinized or suberized walls in the stalk cell to prevent the apoplastic flow of secretory material into neighbouring mesophyll tissue. The histochemical stains also showed that the secretory material present in the glandular trichomes are lipid in nature and essential oils are present. Ultrastructural studies showed polymorphic leucoplasts, few Golgi bodies, numerous vesicles and mini vacuoles, mitochondria and short profiles of endoplasmic reticulum cisternae. Phytochemical tests revealed the presence of essential oils that are terpene-rich. Flavonoids, tannins, saponins, terpenoids, fixed oils and fat, phenolics and cardiac glycosides were also detected in a crude ethanolic extract of the leaves. These chemical compounds appear to be responsible for the medicinal properties for which the plant is traditionally exploited. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

It has been suggested that lipid peroxidation is involved in the loss of seed vigour, although many attempts to examine the relationships between lipid peroxidation and seed vigour have proved equivocal. Studies were undertaken on seed lots of cabbage and soya bean to find evidence for peroxidation by the analysis of i) total and polar fatty acid levels; ii) lipid hydroperoxides; iii) volatile products produced on heating dry seeds; and iv) volatile products produced on imbibition. The loss of polyunsaturated fatty acids (PUFAs) detected in the dry seeds was clearly related to germinability in both soya bean and cabbage seeds. Furthermore, an increase in hydroperoxides was observed in both seed types. Although the relationship of the level of hydroperoxides to germinability was less clear than for the decline in the level of PUFAs, these results suggested that the loss of PUFAs was possibly due to evidence the peroxidation of the seed obtained from the heating lipid, indirect of the seeds suggesting that hydroperoxide breakdown may be necessary in order that the changes in PUFAs become apparent. In contrast to the poor relationship observed between germinability and hydroperoxide level, a marked relationship between hydroperoxide level and seed moisture content was observed in the cabbage seeds. This may be significant with regard to the observed relationship between storability and seed moisture content, although no such relationship was seen in the soya beans. Certain volatile compounds derived from dry heated seeds were related to seed vigour in both seed types and evidence suggests that the lipid hydroperoxides were the source of these compounds. Although the total volatiles counts evolved from imbibing cabbage seeds showed no quantitative relationship to seed vigour, one peak was noted which was clearly associated with the vigour of these seeds. The variability in the volatiles evolved from soya beans on imbibition, however, precluded the detection of any possible relationship between these and seed vigour. In both seed types, results suggest that the volatiles derived on imbibition were of a different source to those derived on heating. A marked increase in the level of hydroperoxides was observed in whole cabbage seeds and soya bean axes of low vigour over the first hour of imbibition. This may suggest that an exacerbation of damage on imbibition was associated with low vigour seeds. In contrast to this, in the seeds of high vigour, the increase in hydroperoxide levels was markedly less or rapidly reduced, suggesting the possible activity of repair mechanisms. Ferrous ions were shown to invigorate both seed types, particularly cabbage seeds. It is suggested that the invigorating effect of these compounds was due to the facilitation of repair, including hydroperoxide breakdown and the quenching of any free radicals. / Thesis (M.Sc.)-University of Natal, Durban, 1986.

Against the background of the increasing pharmaceutical importance of members of the genus Hypoxis L., methods for propagating these plants and for producing hypoxoside (the believed active compound found within Hypoxis species) using in vitro techniques, were investigated. These investigations were accompanied by anatomical observations. Hypoxis rooperi S. Moore was selected as experimental material because of its availability and common usage among researchers studying the genus Hypoxis. Two aseptic procedures were developed for propagating H. rooperi. These being the only procedures as yet to be established and documented, using a member of the family Hypoxidaceae. The first procedure involved the induction of callus and adventitious shoots from flower bud explants of H. rooperi . For this response to be initiated, the buds selected for culture had to be of a specific morphological and physiological age. The best medium determined for inducing a callusing and shooting response from these explants, was a MURASHIGE and SKOOG (1962) medium supplemented with low levels of I-naphthalene acetic acid and high levels of 6-benzylaminopurine. The rate of this response was enhanced by the wounding of flower bud explants (i.e. by the excision of the perianth segments, stamens and style from the buds). Investigations indicated that callus and adventitious shoot formation was inhibited by the acropetal positioning of damaged flower buds on the culture medium. This inhibition was not manifest when buds were placed basipetally or horizontally on the culture medium. Flower bud harvest time was not found to have a marked effect upon the numbers of explants responding in culture. On average 37,5 per cent of the buds formed callus and adventitious shoots throughout the flowering season. The subculturing of callus tissue established from H. rooperi flower buds, onto a MURASHIGE and SKOOG (1962) medium supplemented with the same hormone levels as were initially used to induce callus and shoot formation, resulted in the production of multiple adventitious shoots. Serial subculturing of this tissue indicated that the shoot producing capacity of the callus, was maintained for at least a year. Shoots produced via this method, when inoculated onto a hormone-free culture medium, formed roots. Seventy-five per cent of the plantlets regeneratro in vitro were successfully "hardened-off". Theoretically it was calculated that using the micropropagation procedure developed, almost 81000 H. rooperi plantlets could be established from 100 flower bud explants, within a year. The second aseptic procedure developed, involved the culturing of explants excised from the primary thickening meristem region of H. rooperi corms. The best medium determined for inducing the formation of adventitious shoots from these explants, was a MURASHIGE and SKOOG ( 1962) nutrient solution supplemented with: equivalent low concentrations of I-naphthalene acetic acid and 6-benzylaminopurine; 30 rather than 20 or 40 gl¯¹ sucrose; and 1,0 gl¯¹ casein hydrolysate. Random as opposed to a basal or side positioning of corm explants upon the culture medium, resulted in higher numbers of adventitious shoots being produced. The location of explant excision from within the donor plant was also found to influence shoot productivity. No significant difference was detected in the total number of shoots produced from corm explants harvested at various times of the year. The rooting of shoots differentiated from corm explants posed few problems, as most shooted explants eventually formed roots without being subcultured. Those which did not form roots could be induced to do so, by the inoculation of the shooted explants onto a culture medium either devoid of hormones or containing low I-naphthalene acetic acid levels. Following a rather simple procedure developed, ninety per cent of the plantlets were "hardened-off". From 100 corm explants it was therefore possible to regenerate 104 to 112 plantlets within a 3 to 4,5 month period. Prior to the assessment of the usefulness of in vitro cultures for producing hypoxoside, qualitative and quantitative techniques for detecting hypoxoside, were developed. Using these techniques it was established that only the root-like types of cultured tissue, contained hypoxoside. The levels of hypoxoside detected within these tissues were much lower than those found within mature in vivo grown plants. Using the cultured tissue containing the highest levels of hypoxoside, it was shown that the subculturing of this tissue resulted in a decrease in hypoxoside content. This effect could be overcome by lowering the levels of nitrogen in the medium or by culturing the tissue in the dark. These results showed that the cultured tissue was able to synthesize hypoxoside. To what extent this synthetic rate can be increased remains very much an academic problem and one which deserves more attention. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.