Studies were undertaken to identify the basis of differences in seed coat pigmentation between selected yellow- and brown-seeded Brassicaceae, and near-isogenic yellow- and brown-seeded lines of </i>Brassica carinata</i> (PGRC/E 21164). Histochemical analyses of dissected seed coats from six genera of the Brassicaceae revealed condensed tannin (proanthocyanidin) and phlobaphene pigments in dark-seeded species and in scattered spots in most yellow-seeded species. Anthoeyanins were not detected in any seed coat tissue of these species. In leaf tissue, anthocyanin content was lower in yellow-seeded 'B. carinata' at the three- to four-leaf stage grown at 20 to 25°C, compared to similarly grown brown-seeded plants. At 15 to 18°C, both yellow- and brown-seeded lines produced similar amounts of anthocyanin in the seedling leaves. In TLC and HPLC analyses, 't'-cinnamic acid, dihydromyricetin [trace amounts], dihydroquercetin, dihydrokaempferol and flavonols (quercetin, kaempferol) were more abundant in extracts from seed coats of yellow-seeded ' B. carinata' than in extracts from seed coats of brown-seeded ' B. carinata'. Myricetin was not detected in any seed coat extracts. Mass spectra were determined for phenylpropanoid and flavonoid aglycones from the seed coat extracts and for authentic standards. Dihydroflavanol reductase ('DFR') transcripts from developing seed were absent or less abundant at 5, 10, 20 and 30 days after pollination in the yellow-seeded line compared to the brown-seeded line of 'B. carinata'. 'DFR' transcripts in seedling leaves from the yellow-seeded line grown in warm, bright conditions were less abundant compared to similarly grown brown-seeded material. Seedlings grown at 15 to 18°C produced greater amounts of 'DFR' transcript in both yellow- and brown-seeded lines of 'B. carinata' compared to warm-grown plants. Data from these experiments suggest that seed coat pigmentation in the Brassicaceae is due to condensed tannin and phlobaphene accumulation, not anthocyanins, and that seed coat pigment biosynthesis is down-regulated at dihydroflavonol reductase. The regulatory factor controlling 'DFR' expression in developing seed may be temperature sensitive and also affect anthocyanin biosynthesis in seedlings and in related metabolic pathways: thioglycolic lignin concentration was significantly lower in the yellow-seeded Brassicaccae and in the seed coat tissue of yellow-seeded 'B. carinata' compared to dark-seeded samples.

published_or_final_version / abstract / toc / Ecology and Biodiversity / Master / Master of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

No description available.

No description available.

No description available.

Three studies were conducted to determine the effects of roasting on ruminal degradability of sunflower seeds and the effects of feeding roasted sunflower seeds on ruminal fermentation, nutrient digestibility and milk yield and composition of dairy cows. Experimental treatments were a control diet with no added sunflower seed, a raw sunflower seed diet and a roasted sunflower seed diet. Sunflower seed diets contained 6% fat whereas the control diet contained 3% fat. In study one, two ruminally fistulated cows were used in a randomized complete block design to determine the effects of roasting on ruminal degradation of sunflower seeds. In the second study, three ruminally cannulated lactating Holstein cows were used in a 3 x 3 Latin square experiment to determine the effects of dietary treatments on ruminal fermentation and total tract nutrient utilization. In the last study, three primiparous and six multiparous Holstein cows were used in three 3 x 3 Latin squares to determine the effects of dietary treatments on milk yield and composition.

The conservation of germplasm of indigenous plant species is vital not only to preserve valuable genotypes, but also the diversity represented by the gene pool. A complicating factor, however, is that a considerable number of species of tropical and sub-tropical origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated and metabolically active when shed and cannot be stored under conventional conditions of low temperature and low relative humidity. This poses major problems for the longterm conservation of the genetic resources of such species. Presently, the only strategy available for the long-term conservation of species that produce recalcitrant seeds is cryopreservation. Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The aim of the present study was to develop methods for the cryopreservation of germplasm of this species. Different explant types were investigated for this purpose, viz. embryonic axes (with attached cotyledonary segments) excised from seeds, and two in vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings and adventitious shoots generated from intact in vitro-germinated roots. Suitable micropropagation protocols were developed for all explant types prior to any other experimentation. Before explants could be cryopreserved it was necessary to reduce their water content in order to limit damaging ice crystallisation upon cooling. All explants tolerated dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with survival ranging from 100 – 80%, depending on the explant. In addition, penetrating and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants. The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose and glycerol. Explant survival following cryoprotection and dehydration ranged from 100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic temperatures by cooling at different rates, since this factor is also known to affect the success of a cryopreservation protocol. The results showed that ‘broken’ buds could not tolerate cryogen exposure. This was likely to have been a consequence of the large size of explants and their originally highly hydrated condition. Adventitious shoots tolerated cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached cotyledonary segments were exposed to cryogenic temperatures. In contrast, root production from axes cooled in sub-cooled nitrogen remained high (67 – 87%). Adventitious shoots were subsequently induced on roots generated from cryopreserved axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated roots. In this manner, the goal of seedling establishment from cryopreserved axes was attained. Each stage of a cryopreservation protocol imposes stresses that may limit success. To gain a better understanding of these processes the basis of damage was investigated by assessing the extracellular production of the reactive oxygen species (superoxide) at each stage of the protocol, as current thinking is that this is a primary stress or injury response. The results suggested that superoxide could not be identified as the ROS responsible for lack of onwards development during the cryopreparative stages or following cryogen exposure. The stresses imposed by the various stages of a cryopreservation protocol may affect the integrity of germplasm. Since the aim of a conservation programme is to maintain genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain whether this has been achieved. Thus, explants (axes with cotyledonary segments and adventitious shoots) were subjected to each stage of the cryopreservation protocol and the epigenetic integrity was assessed by coupled restriction enzyme digestion and random amplification of DNA. The results revealed little, if any, DNA methylation changes in response to the cryopreparative stages or following cryogen exposure. Overall, the results of this study provided a better understanding of the responses of germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant types were successfully cryopreserved. Future work can be directed towards elucidating the basis of damage incurred so that more effective protocols can be developed. Assessment of the integrity of DNA will give an indication as to the suitability of developed protocols, or where changes should be made to preserve the genetic (and epigenetic) integrity of germplasm. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.

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. / Thesis (M.Sc.)-University of Natal, 1995.