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.

Diseases caused by bacteria, fungi and viruses pose a significant threat especially to poor rural communities. Viral infections are frequently complicated by secondary bacterial and fungal infections which remain a major challenge globally and in particular, in sub Sahara Africa amongst humans and animals alike. The main aim of this study was to develop a low toxicity plant extract or isolated compound active against viral, bacteria and fungal pathogens from selected plant species. Seven tree species that were investigated were Acokanthera schimperi, Carissa edulis, Ekebergia capensis, Podocarpus henkellii, Plumbago zeylanica, Annona senegalensis and Schrebera alata traditionally used in the treatments of various ailments were selected and extracted using solvents of varying polarity. Extracts of selected plants were tested for activity against two Gram positive and two Gram negative bacterial namely Enterococcus faecalis and Staphylococcus aureus and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli respectively, three fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigates and four enveloped animal viruses: feline herpes virus–1 (FHV-1, dsDNA), canine distemper virus (CDV, ssRNA), canine parainfluenza virus-2 (CPIV-2, ssRNA) and lumpy skin disease virus strain V248/93 (LSDV, dsDNA). The presence of antioxidant constituents in the different extracts and cytotoxicity against three cell types CRFK, bovine dermis and Vero cells were determined. Bioautography and the serial microplate dilution methods were used to determine the number of antimicrobial compounds and antimicrobial activity of extracts against bacterial and fungal pathogens. Virucidal and attachments assays were used to determine the activity against viral pathogens. Qualitative antioxidant activities of extracts were tested using the DPPH reagent and cytotoxicity using the MTT assay. Biological activity was observed in all the extracts against one or more organisms on bioautography. The intermediately polar system (CEF) separated more active constituents. Some extracts had compounds with similar Rf values active against one or more organisms. In both the antibacterial and antifungal assays, acetone extracts had the highest activity followed by DCM against one or more pathogens. Hexanes extracts were the least active. P. henkellii extracts had more active compounds against the bacteria and Annona senegalensisagainst the fungi. In the micro-dilution assay, S. aureus was the most susceptible bacterial organism to extracts of the different plant, followed by P. aeruginosa andEscherichia coli, and E. faecalis the least. C. neoformans on the other hand was the most susceptible fungal pathogen. In the antiviral assay, although activity was observed with hexane extracts of some plants in the virucidal assay, the most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV in the virucidal assay and acetone extracts in the attachment assay. In general the hexane was the least toxic while the intermediate polarity extracts were generally the most toxic indicating that highly polar compounds were possibly poorly or highly absorbed through membranes in the former and later respectively. Of the three cell types used CRFK was the most sensitive followed by bovine dermis and Vero cells the least. Cytotoxicity studies of extracts of the different plants revealed A. senegalensis and A. schimperi extracts were the most toxic plants in the cellular assay. These plants are toxic to animals and the cytoxicity is in line with the in vivo toxicity. The protective effects of antioxidant constituents in some extracts varied and appear to be influenced by the metabolism of the type of cell in culture. It also appears to suggest that metabolism in kidney derived cells can be influenced by species variation in the origin of cells. P. henkellii was selected for isolation of bioactive compound. Three compounds were isolated and their structure elucidated using 13C and 1H NMR and mass spectrometric data. The antibacterial, antifungal and antiviral activity of the isolated compounds 7’, 4’, 7’’, 4’’’, tetramethoxy amentoflavone (C1), isoginkgetin (C2) and Podocarpusflavone–A (C3) were determined. Compound C2 was the most active against E. coli and S. aureus (MIC = 60 ìg/mE) and a selectivity index (SI) value of 16.67. The compound was also active against A. fumigatus and C. neoformans (SI = 33.33) suggesting both antibacterial and antifungal activity with relative safety. Compound C3 had a broad spectrum of activity against E. faecalis and P. aeruginosa with SI values of 4. A less potent activity of the compounds was obtained in both the virucidal and attachment assays against test pathogens, indicating the lower activity of the compounds against tested viral pathogens. The studies further suggest structural activity relationship in the antimicrobial activity of biflavonoids. The compounds C1 and C2 had no toxic effect on the three cell types and mutagenicity studies indicated no activity of these compounds. Podocarpusflavone-A occurs in every species of Podocarpus so far investigated, except P. latifolius. These studies represent the first isolation of bioactive compounds from P. henkellii. Although a different extractant was used than that used by traditional healers, the presence of antiviral compounds in Podocarpus henkelii against two unrelated viruses may justify on a chemotaxonomic basis the traditional use of related species Podocarpus latifoliusand Podocarpus falcatus in the traditional treatment of canine distemper infection in dogs. / Thesis (PhD)--University of Pretoria, 2011. / Paraclinical Sciences / unrestricted