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.

Glutathione transferases (GSTs) catalyse the conjugation of the electrophilic herbicides atrazine, metolachlor, alachlor and fluorodifen with the tripeptide glutathione (GSH). Maize (Zea mays L), contains multiple GSTs with differing substrate specificities which confer tolerance to a variety of herbicides. In contrast far less is known regarding the GSTs in competing weed species. In vivo metabolism studies using seedlings of maize and the weeds Panicum miliaceum. Digitaria sanguinalis, Sorghum bicolor. Setaria faberi. Abutilon theophrasti and Echinochloa crus-galli demonstrated that all species were capable of metabolising radiolabelled atrazine to GSH conjugates and the relative rates of metabolism related well to GST activities. Similarly, GST activities toward atrazine, metolachlor and alachlor correlated well with herbicide tolerance, with GSH availability being less important. GST activities towards metolachlor, alachlor and atrazine were highest in young maize plants and decreased with age, whilst GST activities in S.faberi remained unchanged. At 35 days GST activities were similar in the two species and the atrazine selectivity was lost. GSH content decreased with age in both species. Protein purification studies showed that S.faberi contains 4 GST isoenzymes with differing substrate specificities. The major GST was estimated to account for 0.1 % of die total soluble protein in S.faberi. PCR-amplification of a cDNA prepared from mRNA showed that S.faberi contains a GST with 88% identity to GST I from maize at the nucleotide level and 82% identity at the amino acid level. Similarly antibodies raised to maize and wheat GSTs recognised GSTs in S.faberi. It is concluded that GSTs determine the relative tolerance to chloroacetanilides and atrazine in weed seedlings but may be less important in older plants. The GSTs in S.faberi are similar in complexity to those determined in maize but are expressed at lower levels.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1996. / Includes bibliographical references (leaves 139-161). / Microfiche. / xiv, 161 leaves, bound photos 29 cm

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

Thesis (Ph. D.)--University of Sydney, 2008. / Includes graphs and tables. Includes list of publications: p. iv. Title from title screen (viewed May 5, 2008). Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Chemical and Biomolecular Engineering. Includes bibliographical references. Also available in print form.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 53-54). Also available via the Internet.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

M.Sc. (Biochemistry) / The culture of plant anther tissue in vitro, is an important tool which could be used, when perfectedr in the conventional breeding of crop plants. Using this technique it may be possible to generate bomozygotIc lines in one or two generations in contrast to the seven or eight generations required by conventional breeding programs. Physiological data on the behavior of cultured anther tissue suggests that the genotype is the most Important factor Influencing this factor with respect to wheat barley and most other crop plants. Optimal culture conditions with respect to culture medium and environmental factors have already been established. It would appear that a critical factor, present in the pollen cells of anther tissue, governs the variation and differentiation of the embrionic pollen in the formation of haploid plants. Many important systems of cellular signalling and therefore cellular regulation are mediated by protein kinases and phosphatases. An examination of protein kinase activity in normal and anther tissue may yield information about the process of differentiation. Identification of key kinase activities may provide plant breeders with a means of selecting more responsive genotypes for the use In the anther culture technique In VItro. In this manuscript a caparison is made between protein kinase activities in crude extracts of meristematic...

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N/A / Thesis / Master of Science (MS)