Thesis (MSc. (Botany)) -- University of Limpopo, 2015 / Soybean is an important source of high quality protein and oil for both humans and animals, especially in protein formulations for pharmaceutical and nutriceutical use. This crop is adversely affected by both biotic and abiotic stresses impacting on its productivity. Soybean productivity can be improved via techniques such Agrobacterium-mediated genetic transformation. Soybean is recalcitrant and depends on suitable explants from which new shoots can be regenerated and be amenable for transformation. The goal of this study was to produce transgenic soybean plants that are tolerant to drought stress through Agrobacterium tumefaciens-mediated transformation. Multiple shoot induction on double and single coty-node explants, obtained from soybean seedlings derived from seeds germinated in vitro on Murashige and Skoog culture medium supplemented with cytokinins was studied. The effect of different concentrations of benzyladenine (1.57, 2.00 and 4.00 mg/l), and benzyladenine (2.00 mg/l) in combination with kinetin (1.00 mg/l) was tested. The results show that the double coty-node explants produce the highest number of shoots per explant, an average of 7.93 shoots on Murashige and Skoog medium supplemented with 2.00 mg/l benzyladenine. The lowest number being 1.87 shoots obtained from single coty-node explants cultured on Murashige and Skoog medium containing 4.00 mg/l benzyladenine. The single coty-node explants showed lower frequency (10–57%) of shoot induction when compared to the double coty-node explants (50–83%). The suitability of aminoglycoside antibiotics (hygromycin, tetracycline and rifampicin) for efficient elimination of Agrobacterium tumefaciens after co-cultivation was tested using a well agar diffusion assay. Co-culturing double coty-node explants with Agrobacterium containing pTF 101 vector carrying the Oryza cystatin 1 gene resulted in 76.6, 63.3 and 60.0% shoot regeneration on Murashige and Skoog shoot induction media (shoot induction medium 1, shoot induction medium 2 and shoot induction medium 3) containing hygromycin, tetracycline and rifampicin at 500 mg/l respectively. These antibiotics showed the highest zones of inhibition against pTF 101 using the well agar diffusion assay. On the other hand, 85% plant regeneration was obtained during in vivo transformation following Agrobacterium injection into seedlings. These results imply that vi both in vitro and in vivo protocols were suitable for transgenic shoot regeneration and plant establishment since all the plants continued surviving in the presence of 6.00 mg/l glufosinate-ammonium. Future work will focus on screening of transgenic plants using beta-glucuronidase and isolating the protein encoded by the Oryza cystatin 1 gene to further confirm the generation of transformed plants carrying the gene of interest.

Quality protein maize (QPM) has enhanced levels of two essential amino acids, lysine and tryptophan compared to normal maize. This makes QPM an important cereal crop in communities where maize is a staple crop. The main abiotic factor to QPM production is drought stress. Little information is available on the effect of drought stress on QPM. Therefore, the objectives of this study were to: (i) conduct diversity analysis of QPM inbred lines using morpho-agronomic and simple sequence repeat markers, (ii) screen available QPM inbred lines and F1 progeny for tolerance to seedling drought stress, (iii) determine the combining ability and type of gene action of QPM inbred lines for tolerance to seedling drought stress, grain yield and endosperm modification. The study was conducted in South Africa, at the University of Fort Hare. Morphological characterisation of 21 inbred lines was done using quantitative and qualitative traits. A randomised complete block design with three replicates was used for characterizing the inbred lines in the field. Genstat statistical software, version 12 (Genstat ®, 2009) was used for analysis of variance (ANOVA) and descriptive statistics. Analysis of variance was performed on all quantitative data for morphological traits. Data for qualitative traits was tabulated in their nominal classes. Traits that contributed most to the variation were days to anthesis, days to silking, anthesis-silking interval, plant height, number of kernel rows, ear length and grain yield. Cluster analysis grouped the inbred lines into three main clusters. The first cluster was characterised by tall and average yielding lines, while the second cluster showed the least anthesis-silking interval, and had the highest yield. Cluster three consisted of lines that were early maturing, but were the least yielding. Genetic distances between maize inbred lines were quantified by using 27 simple sequence repeat markers. The genetic distances between genotypes was computed using Roger’s (1972) genetic distances. Cluster analysis was then carried out using the neighbour-joining tree method using Power Marker software version 3.25. A dendrogram generated from the genetic study of the inbred lines revealed three groups that concurred with expectations based upon pedigree data. These groups were not identical to the groups generated using morpho-agronomic characterisation. Twenty one QPM inbred lines were crossed using a North Carolina design II mating scheme. These were divided into seven sets, each with three inbred lines. The three inbred lines in one set were used as females and crossed with three inbred lines in another set consisting of males. Each inbred line was used as a female in one set, and as a male in a second set. Sixty three hybrids (7 sets x 9 hybrids) were formed and evaluated in October 2011, using a 6x8 alpha-lattice incomplete block design with three replicates under glasshouse and optimum field conditions. A randomised complete block design with three replicates was used for the 21 parental inbred lines. Traits recorded for the glasshouse study were, canopy temperature, chlorophyll content, leaf roll, stem diameter, plant height, leaf number, leaf area, fresh and dry root and shoot weights. Data for the various traits for each environment, 25 percent (stress treatment) and 75 percent (non-stress) of field capacity, were subjected to analysis of variance using the unbalanced treatment design in Genstat statistical package Edition 12. Where varietal differences were found, means were separated using Tukey’s test. Genetic analyses for grain yield and agronomic traits were performed using a fixed effects model in JMP 10 following Residual Maximum Likelihood procedure (REML). From the results, inbred lines that were not previously classified into heterotic groups and drought tolerance categories were classified based on their total dry weight performance and drought susceptibility index. Inbred lines L18, L9, L8, L6 and L3, in order of their drought tolerance index were the best performers under greenhouse conditions and could be recommended for breeding new varieties that are tolerant to seedling drought stress. Evaluation of maize seedlings tolerant to drought stress under glasshouse conditions revealed that cross combination L18 x L11 was drought tolerant, while cross L20 x L7 was susceptible. Total dry weight was used as the major criteria for classifying F1 maize seedlings as being resistant or susceptible. General combining ability effects accounted for 67.43 percent of the genetic variation for total dry weight, while specific combining ability effects contributed 37.57 percent. This indicated that additive gene effects were more important than non-additive gene action in controlling this trait. In the field study (non-drought), the experimental design was a 6x8 alpha lattice incomplete block design with three replicates. On an adjacent field a randomised complete block design with three replicates was used to evaluate the parental inbred lines. The following variables were recorded: plant height, ear height, ears per plant, endosperm modification, days to silking and days to anthesis, anthesis-silking interval, number of kernels per row, number of rows per ear and grain yield. General analyses for the incomplete lattice block design and randomised complete block design for hybrid and inbred data respectively were performed using JMP 10 statistical software. Means were separated using the Tukey's test. Genetic analyses of data for grain yield and agronomic traits were conducted using a fixed effects model using REML in JMP 10. The importance of both GCA (51 percent) and SCA (49 percent) was observed for grain yield. A preponderance of GCA existed for ear height, days to anthesis, anthesis-silking interval, ears per plant and number of kernels per row, indicating that predominantly, additive gene effects controlled hybrid performance under optimum field conditions. The highest heritability was observed for days to silking (48.27 percent) suggesting that yield could be improved through selection for this trait. Under field conditions, variation in time to maturity was observed. This implies that these inbred lines can be recommended for utilisation in different agro-ecologies. Early maturing lines such as L18 can be used to introduce earliness in local cultivars, while early maturing single crosses such as L18 x L2, L5 x L9, L3 x L4 and L2 x L21 could be recommended for maize growers in drought prone areas such as the former Ciskei. Single crosses L18xL11, L16xL18, L8xL21 and L9xL6 had good tolerance to seedling drought stress. On the other hand, single crosses L18xL11 and L11xL13 had high grain yield and good endosperm modification. All these single crosses could be recommended for commercial production after evaluation across locations in the Eastern Cape Province. Alternatively they can be crossed with other superior inbreds to generate three or four way hybrids, which could then be evaluated for potential use by farmers in the Eastern Cape.

MSCAGR (Plant Production) / Department of Plant Production / Chickpea (Cicer arietinum L.) is an excellent utilizer of residual soil moisture in agricultural ecosystems. However, its seed quality and hence reproduction is constrained by water stress, seed size and storage conditions. This study was carried out at the University of KwaZulu- Natal (UKZN), Pietermaritzburg Campus. It was conducted to evaluate the performance of chickpea genotypes (Desi-K, Saina-K and ICCV-K) with different seed sizes on seedling emergence (i), seed ageing effect on seed quality and imbibition of genotypes produced under water stressed and non-stressed conditions (ii), and (iii) the effect of water stress during seed development on sugars and protein accumulation, germination and seed vigour. Pot experiments were conducted under glasshouse / tunnel conditions at the Controlled Environment Facilities (CEF). The experiment for objective 1 was laid out as a single factor in completely randomized design (CRD). Data on emergence rate, final hypocotyl and complete emergence was collected. The small seeded Desi-K showed higher and faster emergence compared to medium sized Saina-K and large seeded ICCV-K. In the experiment of the second objective, seeds of the three genotypes were first obtained by production under water stressed and non-stressed growing conditions. They were then aged for 0, 1, 3, 5, or 7 days at 41 ºC and 100% relative humidity to form a 2 x 3 x 5 (water levels x genotypes x ageing) factorial design. Data was collected on germination percentage (GP), mean germination time (MGT), electrical conductivity (EC), tetrazolium chloride test (TZ) and imbibition weight. Seed ageing caused progressive loss of seed viability and vigour in all genotypes, which resulted in lower GP, delayed MGT, reduced TZ staining, cell death and high solute leakage from the seeds produced under the two water regimes. However, the effect was more severe under water stressed conditions. In the experiment for objective 3, seeds of all three genotypes were larger when grown under non-stressed condition compared to those under water stressed condition. These larger seeds had higher seed viability and germination percentage but lower electrical conductivity and mean germination time. Stressed seeds had higher soluble sugars than non-stressed seeds. It was deduced that irrigation during seed development reduces the final sugars and protein content but increases the seed size and physiological quality parameters allied to production of chickpea. Therefore, water provision to chickpea crop is critical during seed development. / NRF