Alfalfa (Medicago sativa) is a valuable forage crop grown throughout the World. While the crop is resistant to attack by many insect pests, it is subject to potentially severe losses through the action of several specific pests, which are adapted to alfalfa as a host. The most economically damaging of these pests is the alfalfa weevil, Hypera postica. This thesis investigates the endogenous defences of alfalfa against insects, which are responsible for its resistance to non-pest species, and develops a strategy for increasing the resistance of alfalfa towards pest species, specifically alfalfa weevil. The role of saponins in the resistance of alfalfa towards non-pest species has been investigated by using successive insect bioassays, carried out with extracts, mixtures of compounds, and purified compounds, to identify which compounds present in alfalfa tissues are responsible for toxicity towards insects. Crude saponin extracts, in 80% methanol, from alfalfa seedling tissues were bioassayed against the cowpea seed weevil, Callosobruchus maculatus. Both extracts from shoot and root tissues caused larval mortality and delayed development when incorporated into an artificial diet at levels comparable to those found in alfalfa, but lower levels of root saponin extracts showed probiotic effects, whereas lower levels of shoot saponins were still toxic. Hydrolysis of the saponins present in these extracts decreased their toxicity. Purified saponin mixtures were prepared by butanol partition and ether precipitation, and were bioassayed against potato aphid (Aulacorthum solani) in a liquid artificial diet, which allowed quantitative effects to be accurately assayed. Shoot saponins showed a concentration-dependent toxic effect, decreasing survival over an initial 5 day period, decreasing growth, and inhibiting fecundity (measured by nymph production) in these insects. Alfalfa root saponins showed no deleterious effects below a threshold level, but caused complete mortality above this level. The alfalfa saponin mixtures were separated into fractions by chromatography on a reverse phase column. Bioassays showed that the toxicity towards potato was associated only with fractions containing saponins, and that fractions containing a component identified as soyasaponin I were more toxic to the aphids than others. Finally, two saponins purified from alfalfa, soyasaponin I and medicoside A, were assayed. These assays showed that soyasaponin was consistently more toxic in effects on mortality, growth and fecundity. It was concluded that alfalfa saponins, and in particular soyasaponin I, were major factors in the resistance of alfalfa towards potato aphid, and other insects. A saponin mixture from another species, sugar beet {Beta vulgaris) was also toxic to aphids, supporting the view that saponins have a general role in resistance to insects. Inhibition of insect digestive proteolysis by expression of a foreign protein protease inhibitor was selected as a strategy to protect transgenic alfalfa against alfalfa weevil. The major protease activity in larvae of this msect was shown to be due to cysteine proteases, which could be inhibited by cystatins. Rice cystatin was produced in large quantity using a recombinant protein expression system in E. coli for use in a "proving" experiment. Incorporation of the rice cystatin into an alfalfa weevil larvae artificial diet decreased survival, showing that this approach was feasible.

This thesis addresses the problem of how to examine a metabolic pathway and identify what are the key elements, specifically with respect to rate-limitation. The aim is to be able to analyze a pathway, identify the bottlenecks and implement genetic modifications to remove these bottlenecks. This is done by defining the system of interest and developing a predictive model using kinetic data. The model predictions can then be verified using fermentation data and genetic techniques to make the appropriate changes for improved performance. The test system chosen for this study was the TOL meta-cleavage pathway for the degradation of benzoate. This system was chosen on the basis of the application of pathway engineering principles to other systems. The modelling strategy and software was developed using principles from metabolic control theory and biochemical systems theory. By applying this to the TOL pathway using kinetic data, the control coefficients for the pathway were obtained as well as the system parameters required for the optimization of the pathway. The simulated results obtained from this model must be validated by experiment. Errors can arise both from incorrect assumptions in the model and from the fact that the kinetic data taken from individual in vitro experiments may not be applicable to the in vivo system. The effect of the presence of the TOL pathway on the behaviour of E.coli JM107 during fermentation was investigated and the transient concentration data necessary to identify the bottlenecks in the pathway measured. This data is then used to calculate the flux control coefficients for the TOL pathway. The predictive results were verified by the fermentation data which identified the first two enzymes in the pathway as having significant flux control coefficients. This final chapter also addresses the issue of flux analysis, that is, the calculation of the fluxes in the system to determine where fluxes to unwanted by-products occur and to indicate points of control. A graphical user interface is used to provide a user-friendly and intuitive means of building and customising metabolic pathways which can then be interfaced with instrumentation to provide on-line flux analysis.

The nodulin-23 gene is an abundantly transcribed soybean gene induced in nodules during symbiosis with Rhizobium. Sequencing of the cDNA and genomic clones revealed one intron within an open reading frame. A 24,275 dalton protein was predicted. The transcription of nodulin-23 gene occurs concomitantly with Lbc$ sb3$ and nodulin-24 genes. The 5$ sp prime$-regions of nodulin-23 and Lbc$ sb3$ genes were sequenced and compared with that of nodulin-24. Three potential cis-regulatory sequences were identified. The presence of trans-acting molecule(s), possibly regulating the expression of these genes, was tested for in vitro by preincubating nuclei from embryonic axes with nodule extract and assaying for gene activation. Nodulin-23, nodulin-24, and Lbc$ sb3$ genes were specifically activated and demonstrated similar kinetics. Several genes used as controls were not stimulated. A nodule factor(s) was shown to bind the 5$ sp prime$-region of nodulin-23 gene. The corresponding DNA regions from the other two coordinately expressed nodulin genes specifically competed for this binding, whereas other genes did not bind this factor at all.

Thesis (M. Sustainability and Technology Policy)--Murdoch University, 2007. / Thesis submitted to the Division of Arts. Bibliography: leaves 179-190.

Thesis (Ph. D.)--Carleton University, 2001. / Includes bibliographical references (p. 179-226). Also available in electronic format on the Internet.

Thesis (M.Sc.(Plant Pathology))--University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (Ph.D.)--Ohio University, November, 2004. / Includes bibliographical references (p. 202-211)

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 142-149). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 113-135). Available also in a digital version from Dissertation Abstracts.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 84-87). Also available on the Internet.