Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiii, 104 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 70-73).

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Plant cells resistant to specific amino acid analogs have been reported to accumulate the corresponding free amino acids. The purpose of this study was to determine the concentrations of fifteen free amino acids: alanine, valine, leucine, isoleucine, glutamate, proline, arginine, aspartate, threonine, methionine, lysine, serine, glycine, tryptophan and phenylalanine in Daucus carota cell lines, resistant either to the proline analog azetidine-2 carboxylic acid (A2C), or to the tryptophan analog 5-methyltryptophan (5-MT), or to both the analogs combined. This study also intended to determine if these analogs influence the biosynthesis of the above-mentioned fifteen amino acids in the cell line resistant to A2C and 5-MT. Carrot cell lines resistant to 5-MT, to A2C, or to both the analogs were selected by incubating carrot cells in liquid growth media containing either 0.3 mM 5-MT, or 0.5 mM A2C for 6 to 16 weeks. Free amino acid concentrations were then determined in the extracts of the cells. Resistance to 5-MT resulted in significant increases in the intracellular concentrations of tryptophan, phenylalanine, leucine, valine, isoleucine, and proline. Resistance to A2C resulted in significant increase in proline only. Resistance to both the analogs caused increases in proline, lysine, phenylalanine, and tryptophan concentrations. In the cell line resistant to both the analogs, the treatment with 5-MT caused increases in leucine, proline, aspartate, threonine, lysine, and tryptophan. The treatment with A2C caused increases in isoleucine, arginine, threonine, methionine, lysine, and glycine, whereas treatment with both the analogs caused increases in threonine, lysine, phenylalanine, and tryptophan. These results indicate the possibility of a common biosynthetic control of a number of amino acids in carrot cells, resembling that found in microorganisms. It is also evident from the results that the analogs play an active role in the biosynthesis of amino acids in the resistant cell lines.

Soyabean meal (SBM), the major protein source in feeds in sub-Saharan Africa, is in short supply. The shortage is a major constraint to intensified animal production to meet increased demand hence the dire need to search for alternatives. Kigelia africana, Mumisops zeyheri, Terminalia sericea and Ximenia caffra are indigenous fruit bearing trees (IFBTs) whose seeds’ potential as alternative protein sources in feeds were evaluated. The evaluation consisted of an initial physico-chemical characterization of the seeds followed by determining in vitro the safety of seed oils on cell lines. Based on the physico-chemical and in vitro evaluation, the most suitable seed was selected, defatted and its meal used as a dietary substitute to SBM in the in vivo trials using adult and weanling male Sprague Dawley rats. The T. sericea seed yield was not viable. Chemically K. africana and X. caffra seed demonstrated potential as protein sources in feeds. M. zeyheri seed demonstrated potential as an energy source. The IFBTs seeds oil yield surpassed that of some traditional oilseed crops. Oleic and linoleic acid were the major fatty acids contained in the oils. In vitro, K. africana, M. zeyheri and X. caffra seed oils suppressed Caco-2 and HEK-293 cell proliferation without causing cell death. X. caffra seed, deemed the most suitable, was defatted and its seed meal used in the in vivo trials. In mature rats, dietary substitution of SBM with the defatted X. caffra seed meal did not affect (P > 0.05) dry matter intake, apparent digestibility of nutrients and nitrogen absorption and retention. In weanling rats, the defatted X. caffra seed meal had no effect on termination (body mass at the end of the feeding trial) and empty carcass mass and linear growth of the rats. Metabolic substrate storage, fasting blood glucose concentration and the general health profile of the growing rats were not altered by dietary X. caffra seed meal. The defatted X. caffra seed meal increased the mass of the stomach and small intestine (P = 0.0071; P = 0.0001) of rats on the test diet where a 100% dietary crude protein (CP) from SBM was substituted by CP from the defatted X. caffra seed meal. Defatted X. caffra seed meal could substitute SBM in rat and possibly monogastrics feeds without compromising digestibility, nitrogen balance, growth and general health.

Thesis (M. Phil.)--University of Hong Kong, 1985.

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published_or_final_version / Botany / Master / Master of Philosophy

In vitro cell and tissue culture systems were developed for sugarcane in order to utilise current transformation techniques to introduce genes to South African sugarcane varieties, which would be difficult, if not impossible to achieve in conventional breeding programmes. Embryogenic calli were initiated in the dark from stem explants of sugarcane varieties NCo376 and N13, on a MS medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1 g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the somatic embryos were cultured in a light/dark photoperiod for a further 2 months. The best combination of sucrose and 2,4-D for callus initiation, and subsequent plant regeneration, was 20 g/l and 2 mg/l, respectively. Plant yields ranged from 16 to 36 plants per gram fresh weight callus, and the yields were not significantly increased by the addition of activated charcoal to the regeneration medium. When plantlets reached a height of 10 cm, they were transferred to autoclaved soil in pots, hardened-off and placed in the glasshouse. Suspension cultures were initiated from friable NCo376 calli in liquid MS medium shaken at 100 rev/min in the dark at 27°C, and were subcultured every 3-7 days. Protoplasts from various sources (leaf, calli and suspension cultures) were obtained after enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2 g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium and magnesium salt solution. Protoplasts cultured for 48 h resulted in a loss in viability of 84%. The potential of the seed as a recipient for direct gene uptake was investigated, as this eliminated the need for in vitro culture and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was demonstrated, and seeds with the testa removed exhibited higher initial uptake rates than those with intact seed coats. However, transient expression, using the GUS reporter gene (coding for bacterial B-glucuronidase) carried on plasmid pBI221, could not be conclusively shown using the histochemical GUS assay, due to GUS activity generated by either microbial contamination or endogenous plant GUS activity. Neither microwaving to eradicate contaminants nor the addition of methanol (20%) to the GUS incubation buffer were successful in overcoming positive results observed in control seeds. An alternative approach to sugarcane transformation, using PEG-mediated DNA uptake and subsequent transient expression of GUS by protoplasts was investigated, but microbial contamination was a persistant problem and no positive results were observed. Further examination and elimination of endogenous contamination is required before transformation studies can be continued. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur. / The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%). / The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass. / The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

Tissue culture studies were carried out on two ornamental plant species, Strongylodon macrobotrys A. Gray and Peperomia clusifolia Hook. Successful in vitro regeneration of plantlets was achieved in the latter species, using leaf and stem explants. The basal medium of Murashige and Skoog (1962), in combination with various levels of NAA and K, was utilised. Strongylodon proved refractory to both establishment of a thriving callus culture and in vitro formation of roots and shoots. Several media were utilised, Miller's (1963) medium proving the most successful for the production of callus. Different combinations of the growth regulators NAA, IBA, BA and K were used to determine optimum levels of these substances for callus production. Root/shoot induction studies were carried out using the basal medium of Miller (1963) plus various concentrations of IBA in combination with K or BA. Alternatively,the basal medium was used without added growth regulators. Internodes, nodal segments, leaves, pulvini, flower parts and seeds were used in the study. No plantlets were obtained from Strongylodon explants. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1982.