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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.

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Since the early 1990s, the concept of substantial equivalence has been a guiding principle of the Canadian Food Inspection Agency and Health Canada's regulatory approach toward products of plant biotechnology destined for the food and livestock feed markets. To assess substantial equivalence in terms of chemical composition, genetically modified (GM) plants are compared to conventional counterparts at the level of macro- and micro-nutrients, allergens and toxicants. Such targeted comparative analyses are limited in their scope and their capacity to detect unintended changes in chemical composition. There is a need to develop more effective testing protocols to improve the substantial equivalence assessment of GM crops. The objective of this thesis was to explore high-density oligoarrays as tools to assess substantial equivalence of Roundup Ready(TM) soybean. Three conventional and two GM soybean varieties were selected according to the similarity of their performance in field trials. Total RNA was extracted from first trifoliate leaves harvested from soybean plants grown in a controlled environment until the V2 stage. To annotate the 37 776 soybean probesets present on the multi-organism Soybean Affymetrix GeneChip(TM), consensus sequences were aligned with TIGR Soybean Gene Index tentative consensus sequences using BLASTN. After redefining the chip description file to exclude non-soybean probesets, the effects of three different normalization methods (Robust Multichip Average (RMA), Microarray Analysis Suite (MAS 5.0) and Model-Based Expression Index) were compared and Significance Analysis of Microarrays (SAM for R-Bioconductor) was applied to detect differential gene expression between conventional and GM soybean varieties. Eleven candidate genes were selected for further studies.

Plants have evolved numerous strategies for the adaptation to drought. Although many investigations reported on the potential value of proline accumulation during environmental stress, it is still unknown whether or not a constitutive higher level of proline accumulation enhances plant tolerance. Thus, it was investigated if underproduction and overproduction of proline will influence the susceptibility to drought stress in soybean plants. This was made possible with the transformation of soybean plants with an L-Δ¹-pyrroline-5-carboxylate reductase (P5CR) gene. First, an Agrobacterium-mediated vacuum infiltration transformation system, using partially germinating Carnia 2233 soybean seed, was established through the assessment of several conditions that can affect transformation efficiency with the use of β-glucuronidase reporter genes. Transformation was confirmed with PCR and Southern blot analysis and results indicated that stable transgenic soybean plants were obtained within one generation with a transformation rate of± 30%. This technique was used in the transformation of Carnia 2233 soybean seed with the P5CR gene in the antisense orientation under the control of an inducible heat shock gene promoter (IHSP). It was confirmed that the P5CR-IHSP gene construct was integrated into the soybean cells and was conserved over three generations. Physiological screening of the antisense P5CR transgenic plants in the greenhouse proved that, with activation of the promoter, an under-expression of the P5CR gene and subsequent inhibition of the accumulation of proline were experienced during drought and osmotic stress. The decline of the viability of the transgenics with prolonged drought stress, as monitored with a woodenbox screening test, is an indication that proline is needed for survival of soybean plants under drought stress conditions. The transgenic plants demonstrated a sensitive reaction in contrast to the control plants that displayed a tolerant reaction to osmotic stress in a TTC assay. The underexpression of the P5CR gene resulted in a decline protein synthesis due to proline shortage as was observed with the evaluation of the efficiency of protein synthesis. All these results suggest that a decrease in the proline level due to the antisense P5CR gene, yielded plants that are more osmotic and drought stress sensitive. Subsequently, the soybean cultivar Ibis was successfully transformed with the P5CR-IHSP construct in the sense and antisense directions in order to test the reproducibility of the transformation process and to assessed the link between the biochemical traits involved in the drought stress mechanism. Three different experiments were conducted: a mild heat and drought stress on "To" transgenic plants exploring changes in chlorophyll fluorescence transients, a mild heat stress on "T1" transgenic plants comparing proline accumulation and chlorophyll fluorescence transients and a severe drought and heat stress on the "T1" transgenic plants comparing proline accumulation NADP⁺synthesis and chlorophyll fluorescence transients. Chlorophyll fluorescence transients were successfully used as a screening method for transgenic soybean plants during this study. The sense transgenics responded to the mild stresses with a significant decrease in their electron transport, trapping and absorption compared to the antisense plants that displayed significant increases in electron transport and trapping. During the severe stress, the antisense transgenics experienced total photoinhibition indicated by the enormous loss of electron transport but the sense plants had the ability to overcome the stress as is revealed in the increase in the electron transport. It was demonstrated that although proline accumulation yielded no significant differences during the mild heat stress, the sense plants accumulated substantially more proline than the control and antisense plants during the severe heat and drought stress. It was demonstrated that proline plays an important role in the plant's response to a drought stress as well as in the recovery phase after drought, as the sense plants also had the ability to reduce the accumulated proline during the recovery period in contrast to the antisense transgenics that experienced protein degradation. The transgenics responded to a period of heat and drought stress with a reduction in NADP⁺ levels in the antisense plants and increasing levels in the sense plants. The sense plants were able to fully recover after the stress period, thus adaptation to drought may depend on different mechanisms, including the capacity to maintain high levels of proline and to regenerate them through the "reduction" of NADP⁺. It was possible to alter the drought tolerance of Ibis by transformation with antisense and sense P5CR gene constructs, which resulted in respectively more sensitive and more tolerant Ibis plants. It can be concluded that over-expression of P5CR during a drought stress resulted in higher proline levels, better photosynthetic efficiency, higher NADP⁺ production and thus a more drought tolerant plant. This study gave additional proof that a constitutively higher level of proline accumulation enhances drought tolerance in soybean. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

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