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.

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Technikon Natal, 2002. / Lack of suitable techniques for field collection of the germplasm of different species, and spoilage of samples in transit, hinder efforts to collect, conserve, distribute and regenerate most plant germplasm in vitro. The aims of this investigation, therefore, were to address problems encountered in collection of field germplasm from species and hybrids of Eucalyptus (TAG5, TAGI4, ZG14, GC550 and GU2IO) that are propagated vegetatively and Trichilia dregeana, which has recalcitrant seeds. Simple in vitro culture-based protocols were developed to minimise contamination and maintain viability of plant material for sufficient time for it to be transported from the field to the tissue culture laboratory. From the two simulations of 48 h 'transportation' conditions for explants of Eucalyptus species investigated, those in bottles containing sterile vermiculite exhibited no contamination and greater than 50% bud break, regardless of whether or not field surface sterilization with alcohol had been done. In contrast, when explants were enclosed in cling wrap, contamination was high and bud break levels low. For selection of the more suitable Eucalyptus explant, two types were investigated: nodal explants each with one half leaf (type 1) and stem segments with three nodes (type 2). As type 2 showed considerably better shoot yields (up to 1624 shoots per 100 explant), and were more practical to use with respect to space, such trinodal stem segments were deemed best for collection. Of the sterilization procedures investigated, treatment with 70% (v/v) alcohol prior to storage was found to be most suitable in almost all cases. For plant material with high endogenous microbial contamination, the bud break medium was supplemented with Benomyl and calcium hypochlorite, each at 0.5 and 1.0 g.r). Alcoholtreated, stored explants cultured on bud break medium with 1.0 g.r) calcium hypochlorite exhibited low levels of contamination and an increased final yield (up to a maximum of 930 shoots per 100 explants). Thus, this protocol was employed for field material of E. grandis clones TAG5, TAGI4 and ZGI4. For these clones, stored type 2 explants / M

Indigenous communities have developed a wealth of knowledge, which plays a crucial role in providing leads for the use of genetic resources and bioprospecting. However, such knowledge is under increasing threat due to the misappropriation of the biological resources and associated traditional knowledge of indigenous communities, through both bioprospecting, as well as the inappropriate exercise of intellectual property rights. The internationally agreed Convention on Biological Diversity (CBD) attempts to provide a bulwark against biopiracy and although it assists indigenous communities to regain some control, the CBD has proven inadequate in the protection of the traditional knowledge of indigenous communities. The subsequent Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (Nagoya Protocol) attempts to address some of these limitations but unfortunately has its own shortcomings, as it was largely concluded on the basis of a compromise between developed and developing countries. This dissertation will undertake a critical analysis of the provisions of the CBD and Nagoya Protocol, with a view to establishing the level of protection these instruments afford indigenous communities. It will be shown that notwithstanding the drawbacks of both the CBD and Nagoya Protocol, they nevertheless represent major achievements in the journey to protect the genetic resources and associated traditional knowledge of indigenous communities. It is in this context that this dissertation will analyse South Africa’s Access and Benefit Sharing (ABS) regime in relation to the protection it affords indigenous communities and in the light of the implementation challenges that such legislation presents. A particular focus will be on whether South Africa’s ABS legislation complies with the country’s international obligations relating to the protection of indigenous communities and whether South Africa’s approach to the protection of the genetic resources and associated traditional knowledge of indigenous communities, in the context of bioprospecting, is adequate or whether there exists potential for its enhancement. / Thesis (LL.M.)-University of KwaZulu-Natal, Durban, 2013.