Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2003. / South Africa produces 190 000 tonnes of soybean per annum. Seed producing companies require knowledge of the diversity of the germplasm to produce hybrids that will be competitive in local and overseas markets. Furthermore, they need to ascertain the presence/absence of the anti-nutritional factor, Kunitz trypsin inhibitor protein. Currently, seed producing companies plant the seed and wait for the grow-out in order to select desirable traits. This process is time-consuming, tedious and does not necessarily ensure the selection of the best genetic stability as it is based on phenotypic expression alone. This study was undertaken to evaluate a molecular method to determine the genetic diversity among soybean parent lines and optimize a method which can be used to evaluate seeds for the Kunitz trypsin inhibitor protein / M

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.

A study was conducted to determine the responses of 10 selected soybean (Glycine max L.) genotypes to potassium silicate (KSi) and Trichoderma harzianum (Eco-T®) applications. Preliminary studies involving two independent experiments were conducted under controlled conditions at the University of KwaZulu-Natal during 2010. Potassium silicate at three concentrations (0, 200 and 250ppm) were applied twice weekly over a period of four months to the genotypes laid out in a randomized complete block design. Subsequently, a field experiment was conducted at Ukulinga Research Farm of the University of KwaZulu-Natal, Pietermaritzburg during 2010/2011 to investigate the responses of the genotypes to KSi at 0 and 200ppm, with and without(Eco-T®) seed treatment. This experiment was set out in a randomized complete block design with three replications. Data collected included number of days to 50% flowering, number of days to 50% maturity, plant height, number of pods per plant, number of seeds per pod, 100 seed weight, root mass, shoot mass, seed yield and harvest index. The total number of root nodules formed and the number of active nodules were determined at end of the field experiment. In most cases a decrease was noted in total nodule formation as well as a decrease in the number of active nodules that formed. In the controlled environments there was a significant interaction between genotype and KSi concentrations for all measured traits. In most cases KSi applied at 200ppm was more successful in enhancing growth, improving seed yield and resulted in high harvest indices. The genotypes that produced the highest seed yield and harvest index in these environments were Williams and Barc-2 at 200ppm KSi. Results from correlation analysis revealed that harvest index and seed yields were generally positively associated with plant height, number of pods per plant and 100 seed weight, which in turn were the traits that contributed to most of the variation to seed yield and harvest index as revealed in the principle component analysis (PCA). The field experiment revealed a significant interaction between genotype x KSi x Eco-T®. Potassium silicate applied at 200ppm with Eco-T® usually promoted growth, seed yield and high harvest indices for all the genotypes. The PCA showed seed yield and harvest index were the traits that contributed to most of the variation. Genotypes Williams, LS6161R, Magoye and Barc-2 were the best seed yielders with the highest harvest indices that responded strongly to the combined use of KSi and Eco-T® under field conditions. Genetic comparison of the ten soybean genotypes with eight microsatellite markers revealed the close genetic relationship between Williams, LS6161 R and Magoye. A link between Barc-2 and Williams was noted by the common parent Clark. Therefore, for these genotypes, the application of KSi at 200 ppm with Eco-T® under field conditions effectively increased seed yield, ranging from 0.45 to 65.26% for some genotypes when compared to the control. An increase was also noted for other agronomic traits and harvest index. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.