M.Sc. / The family lridaceae includes genera such as Sparaxis, Freesia and Babiana which are popular flowering plants. The purpose of this study was the propagation in tissue culture of Sparaxis plants. After the applicable techniques have been formulated, the aim was to test the efficiency of the techniques on the other genera. The techniques can then be extended to endangered genera. Apical meristems as well as axillary meristems were dissected aseptically and grown on the medium of Heinz and Mee (1969). It is difficult to acquire aseptic explant material of Sparaxis because of the net-like fibres surrounding the corm. Growth of aseptic meristem explants occurred within a week under 16h light and 25 ± 1 °C. Proliferation of meristems occurred and gave rise to more than one plant from the same explant. The study was extended to seeds as an explant source, as well as the influences of various factors such as pH, carbon source and gelling agents, on the growth and proliferation of the plants. Rooting of the plants was investigated and hardening done for transfer of the plants to the greenhouse.

No description available.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide. / CSIR

We have studied the effects of exogenous salt on whole plant and suspension culture cells of the halophytic tomato Lycopersicon pennellii. Under low salt conditions (2.9 dS/M) plants showed enhanced (halophytic) growth (107% of control). At moderate (7.5 dS/M) and high (18.5 dS/M) salt levels, salt stress reduced growth to about 78% and 40% of control respectively. Salt-induced changes in root mRNAs were analyzed via two-dimensional PAGE of cell free translation (CFT) products. We have identified 14 proteins whose levels were enhanced by exogenous salt. One of these proteins was unique to low salt induced halophytic growth. This system allowed for discrimination between proteins up-regulated at all salt levels and those up-regulated only during salt stress induced growth reduction. Ten proteins were identified whose levels were reduced by exogenous salt. Once again, one could identify a subset of proteins whose levels were reduced only under salt stressed conditions. Proteins identified in this study are candidates for roles in growth maintaining stress adaptive metabolism in L.pennellii. These data underscore the complexity of the genetic control of salt metabolism in higher plants. The effects of exogenous salt on protein synthesis and accumulation were studied in suspension cultures of L.pennellii. Two salt levels were applied to the cells. Under low salt conditions (LS, 10 mM), L.pennellii cells showed enhanced (halophytic) growth. Under high salt conditions (HS, 50 mM), the cells showed reduced (salt-stressed) growth. Changes in proteins with time were analyzed by a combination of cell free translation, in vivo labeling and total accumulated protein. In vivo labeling studies showed that the pattern of steady state protein synthesis was disrupted shortly after addition of salt. High salt induced greater disruption in the pattern. Over time, the steady state levels of most proteins shifted back towards those of the unstressed-control. However, the level of several proteins remained altered. Analysis of proteins whose levels increased with exogenous salt showed differences in the response patterns that may allow for discrimination between proteins involved in growth maintaining and stress shock responses.

No description available.

Paclitaxel is an anticancer natural product with several biomedical applications produced by Taxus species, with a demand exceeding its supply. We have developed cambial meristematic cells (CMCs) from Taxus cuspidata as high yield source of paclitaxel. The biosynthesis of paclitaxel is predominantly under transcriptional control. Thus, the identification of transcriptional regulators of paclitaxel biosynthesis and their subsequent manipulation may enable further yield enhancement in Taxus CMCs. Previously, Roche 454 sequencing was employed to establish the transcriptome of T. cuspidata CMCs treated with the plant immune activator methyl jasmonate (MeJA). The bioinformatic analysis identified 19 jasmonate related transcription factors (TFs), based on their differential expression. Results of the Arabidopsis thaliana transient assay screen identified two MYB TFs that constitute positive regulators for paclitaxel genes, named MYB3 and MYB4. In this thesis, MYB3 and MYB4 showed in vitro binding to the cis-elements in ten promoters of paclitaxel genes using the electrophoretic mobility shift assay (EMSA). Then, a Taxus CMC protoplasts transient assay demonstrated that the expression of MYB3 and MYB4 trans-activated all tested genes. Further, MYB4 was found to activate the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) gene, key in the mevalonate pathway and precursor of paclitaxel biosynthesis. MYB3 and MYB4 were capable of auto-regulating their own transcription, constituting an important control point for paclitaxel biosynthesis. A possible mechanism for the early activation of MYB3 and MYB4 after MeJA elicitation is proposed. Finally, preliminary results on the expression of MYB3 and MYB4 in unelicited T. baccata CMC protoplasts indicate that their transient expression was sufficient to increase accumulation of paclitaxel and the precursor, 10-deacetyl baccatin III, highlighting their utility for paclitaxel production.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving . Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5.0% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1.0% activated charcoal, added before autoclaving . In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolysed The adsorption of 2, 4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal from methanol and aqueous solutions was determinated using HPLC. The amount of the added 2,4-D decreased in both methanol and aqueous solutions in the presence of activated charcoal, compared with those in the absence of activated charcoal. In methanol and aqueous solutions, activated charcoal used at the level of 0.1% significantly reduced 2,4-D. About 68.4% and 60.9% respectively of the added 2,4-D was adsorbed by activated charcoal (1.0%) from these solutions. The changes of inorganic elements in MS-salt solutions, in the presence of activated charcoal, were analysed by SEM-EDX. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe) and zinc (Zn) deceased in the presence of activated charcoal, while the concentrations of potassium (K), copper (Cu), manganese (Mn), phosphorus (P), and sulphur (S) increased in the MS salt solution in the presence of activated charcoal compared with no activated charcoal in the medium. This suggests that activated charcoal adsorbed calcium, magnesium, iron and zinc and released copper, manganese, phosphorus and sulphur. Rooting occurred when 7-day-old seedling hypocotyls of Daucus carota L. Cape Market were placed on MS medium supplemented with 2,4-D, and IAN/NAA in the presence of activated charcoal. Hypocotyls did not produce roots on the 2,4-D containing media in the absence of activated charcoal. The roots were produced polarly on the NAA/IAA-containing media in the presence of activated charcoal. No-polarity of root formation was observed on media supplemented with NAA/IAA without activated charcoal. Different responses of hypocotyls to a series of 2,4-D concentrations (0.5, 1.0, 3.05.0, 8.0, and 10.0 mg l ¯¹) were observed on media supplemented with 0.02, 0.1 and 0.5% activated charcoal. In the NAA/IAA containing media in the presence of activated charcoal, root number per hypocotyl decreased. Root number perhypocotyl, on the media supplemented with NAA and IAA, increased when hypocotyls were pre-cultured on MS medium supplemented with 2,4-D (1.0 mg l ¯¹) for 2-3 days. When hypocotyls were pre-cultured on a 2,4-D containing MS medium for 5 days, embryos emerged from the hypocotyls directly on the medium supplemented with 2,4-D in the presence of activated charcoal. Addition of activated charcoal to MS medium supplemented with 2,4-D resulted in somatic embryogenesis of Daucus carota. Somatic embryos were not formed on the medium in the absence of activated charcoal. In suspension culture, the incorporation of 0.01 to 1.0% concentrations of activated charcoal to the MS medium, irrespective of 2,4-D, increased the number of somatic embryos produced. The maximum number of somatic embryos were produced with 1.0% activated charcoal. Further development of embryos of Daucus carota occurred on the media in the presence of activated charcoal, and the embryos subsequently regenerated normal plantlets. Abnormal somatic embryos followed the addition of 3.0% activated charcoal to the medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Hybrid clones of the fast-growing Eucalyptus grandis and cold-tolerant E. nitens (GN clones) have been identified by the South African Forestry Industry as being highly suitable for plantations in cold-dry marginal areas. However, one of the main problems regarding their propagation is the difficulty in rooting of cuttings, both in vitro and ex vitro. The aims of this investigation, therefore, were (1) to develop widely applicable and efficient in vitro rooting system(s) for these commercially important clones, and (2) to assess some physiological characteristics of the roots produced. Adventitious shoots (15-20 mm in length) were obtained (l0 shoots/explant) from axillary buds on Murashige and Skoog's (MS) medium containing 0.01 mg.l-1 NAA, 0.01 mg.l-1 IBA and 0.2 g.l-1 FAP. The effect of various medium components, as well as modification of culture environment on in vitro rooting, were investigated. The highest rooting frequencies in clones GN121 (75%) and GN107 (65%) were achieved on l/4 MS with additional 0.22 g.l-1 CaCl2..2H2O and 0.18 g.1-1 MgS04.7H2O, 0.1 mg.l-1 IBA, 0.1 mg.l-1 biotin, 0.1 mg.l-1 calcium pantothenate, 15 g.1-1 sucrose and 4 g.l-1 Gelrite. Best culture conditions were an initial 72-hours dark incubation followed by a 16-hours day/8-hours night photoperiod at a PPFD of 37 µmol.m-2.s-1 and 23°C day/21°C night for seven days, after which the PPFD was increased to 66 µmol.m-2.s-1 at 27°C day/21°C night for 18 days. Towards the development of a more widely applicable in vitro rooting protocol for GN clones, the use of Agrobacterium rhizogenes strains was investigated. Production of transgenic roots was observed on carrot discs and shoots from seedlings of Eucalyptus grandis and E. nitens, but not on shoots of GN clones. Therefore, a method needs to be established for the successful transfer and integration of the Ri plasmid of Agrobacterium into the hybrid plant genome for induction of transgenic roots. The quality of roots produced in vitro and from cuttings was assessed by examination of root anatomy and hydraulic characteristics. Adventitious roots were prepared for measurement of hydraulic conductivity by detopping explants, then filtered, acidified distilled water was drawn through undisturbed potted root systems under partial vacuum, causing no damage to the roots. Initial studies showed that tissue culture-derived roots exhibited a higher specific root mass hydraulic conductivity than those derived from cuttings (6.46 x 10-6 vs. 3.06 X 10-6 g.kPa-1.s-1.g-1 dry root), probably due to root architecture. Curves relating vulnerability to water potential were constructed and both types of roots showed vulnerability to cavitation at high water potentials. Differences were also observed in staining reactions (safranin and fastgreen) which might suggest differences in presence and level of secondary metabolites in these roots at the juvenile stage. Applications of the developed protocols and future research strategies are discussed. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18421号 / 生博第301号 / 新制||生||40(附属図書館) / 31279 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 佐藤 文彦, 教授 永尾 雅哉, 教授 福澤 秀哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM