The objective of this study was to develop and implement an advanced generation breeding programme at the Institute for Commercial Forestry Research (ICFR) to manage and integrate the many and disjunct breeding and production populations of Eucalyptus nitens established by various entities over the past 30 years at multiple sites in South Africa. To develop such a breeding strategy, a good understanding of the population genetics, and the underlying assumptions made by tree breeders about the species, was needed. Eucalyptus nitens is an important forestry species grown for pulp and paper production in the temperate, summer rainfall regions of South Africa. A tree improvement programme has been ongoing at the ICFR for three decades. The measurement and statistical analysis of data from eight F1 trials established during the 1980s and 1990s have enabled characterisation of the ICFR’s breeding population. Provenance testing showed that the more northerly New South Wales (Australia) Eucalyptus nitens provenances of Barren Mountain and Barrington Tops are distinctly better suited to growth in South Africa than the southern New South Wales provenances and the Victorian provenances, Penny Saddle and Bendoc. Generally, the species was not badly affected by Coniothyrium canker. High Type B genetic correlations for all sites pairs, except one comparison, ranged from 0.75 to 0.99 for diameter at breast height at 76 to 113 months, indicating very little, or no, genotype by environment interaction for diameter at breast height for the genotypes tested in the F1 generation. Narrow sense heritability estimates ranged from 0.01 to 0.34, indicating that the species provides a breeding opportunity for improvement of diameter growth. High genetic correlations of greater than 0.90 between diameter measurements at 52 to 62 months after establishment and diameter measurements at 94 or 113 months were found, indicating that selections can be made reliably at five or six years. Diameter measurements at both 60 months and full rotation (94 to 113 months) were highly correlated with the final height measurements in these trial series (rg > 0.71 and > 0.83, respectively). Predicted genetic gains for the F2 over the F1 generation were highest in the trials at Goedehoop and Arthur’s Seat, with predicted increases in diameter at breast height of 3.07 cm (17.1%) and 3.17 cm (20.7%), respectively, at full rotation. Genetic improvement in the species has been slower than anticipated due to delayed and infrequent flowering and seed production. Three genetic gain trials were established, firstly, to quantify the gains that have been made in the first generation of improvement in the breeding programme; and secondly, to establish whether a number of seed source and orchard variables influence the performance of the progeny. These variables were: the number of flowering trees in the seed orchard, year of seed collection, seed orchard origin and composition of seed orchard seed bulks. Diameter at breast height and tree height were measured in the trials at between 87 and 97 months after establishment, and timber volumes and survival were calculated. Improved seed orchard bulks performed significantly better (p < 0.01) than unimproved controls in the field trials, and genetic gains ranging from 23.2 to 164.8 m3ha-1 were observed over the unimproved commercial seed. There were significant differences (p < 0.01) in progeny growth between the levels of flowering, with higher levels of flowering (R 40 %) producing substantially greater progeny growth than lower flowering levels (S 20 %). The seed orchard origin had no effect on progeny growth in this trial series. This suggests that seed collected from any of the four seed orchards tested will produce trees with significant improvement in growth. Various scenarios investigating a range of assumptions were developed and used to predict genetic gain in the F2 populations. These were compared with realised gains achieved in the genetic gain trials. The family nested within provenance scenarios proved to be closer to realised gain than the family across provenance predictions. Two scenarios were used for family nested within provenance: Firstly, actual flowering for family nested within provenance; and secondly, estimated flowering after a 30% roguing of poor families. For both scenarios, a coefficient of relationship of 0.33 predicted gains closest to the realised gains. Indications were that the effects were additive, and that very little or no heterosis had occurred. The statistical information suggested that outcrossing in the seed orchards was > 80%. This study provides an objective and quantitative assessment of the underlying assumptions used for estimating genetic parameters, and predicting gain in this population of Eucalyptus nitens. At the same time that genetic gain trials were established, F2 trials were planted, using seedlots collected from F1 seed orchards. Analysis of the two F2 trials showed that realised gains for diameter at breast height at 87 months were close to the predicted values and ranged from 1.02 cm to 1.90 cm. Two exceptions were the sites at Helvetia and Babanango, where gains were under- and over-predicted, respectively. Realised heritability estimates, which are related directly to the realised gain and the actual selection intensities used in the seed orchards, reflected this trend. Estimation of breeding values allowed for selection of elite individuals in top families. Both grand-maternal provenance origin and F1 maternal effects were significant in the F2 trials. A Type B genetic correlation of 0.61 for diameter at 87 months indicated the possible presence of genotype by environment interactions for the two F2 sites. A low narrow sense heritability estimate of 0.06 for diameter at breast height at 87 months at one F2 site indicated that more emphasis should be placed on family information rather than individual information at this site. A heritability estimate of 0.17 for diameter at breast height at 87 months at the second site, however, indicated that further improvement is possible in this population of Eucalyptus nitens. Modelling of predicted genetic gain using various breeding strategy scenarios can be a useful tool in assisting with the decision on which strategy or management plan will deliver the most genetic gains per unit time. Such modelling, using the parameters established in the first part of the study, played an important role in developing the advanced generation breeding strategy for Eucalyptus nitens. In addition, the modelling exercise highlighted various management options which could be used to increase gains in the existing production populations or orchards. Indications are that additional roguing of 1) existing Clonal Seed Orchards based on results of F2 trials (i.e., backward selection); and 2) F1 Breeding Seed Orchards based on stricter provenance selection, will markedly increase the quality of the seed produced from these orchards within one season. This study also highlighted the importance of shortening the breeding cycle in Eucalyptus nitens, particularly in view of the delays caused by reticent flowering and seed production in the species. The information and understanding gathered from this study led to the development of a proposal for an advanced generation breeding strategy in Eucalyptus nitens. This proposal uses parental reconstruction of open-pollinated progeny to secure pedigree information of forward selections, thus combining the benefits of increased genetic gain with a shortened breeding cycle. Recommendations on the management and adaption of current production populations to increase gains have been made, because establishment and management of improved material in seed orchards to ensure a sustainable supply of improved seed to the South African forestry industry, is a key objective of the ICFR Eucalyptus nitens breeding programme. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

M.Sc. / The 11B/10B stable isotope ratio in wine- and provenance soil samples of four South African wine regions was determined by means of quadrupole inductively coupled plasma mass spectrometry (ICP-MS). The data obtained was combined with multi-element results for the same samples. Discriminant analysis and ANOVA statistics were utilized to create a footprint of wines compared to their provenance soil. The results showed that the stable boron isotope ratio was a distinguishable characteristic in wine and soil samples for the Stellenbosch, Robertson, Swartland, and Walker Bay wine regions. The potential improvement of precision and accuracy was investigated specifically for ICP-QMS (quadrupole mass spectrometry) analysis of boron isotope ratios. Sample preparation methods for the matrices used in this work were developed and applied. The influence of the matrix components was investigated. Ion exchange separation procedures were developed for the separation of total B from the wine and soil matrices. Results were treated mathematically to minimize mass bias effects. It was found that combined matrix effects and TDS (total dissolved solids) deposition did have a pronounced effect on the 11B/10B isotope ratios of the samples. The possibility of boron isotope fractionation was investigated in vine plants. Hydroponic experiments were set up with test plants of two cultivars. A 10B enriched nutrient solution or growth solution with natural 11B/10B ratio was supplied to the plants during alternating periods of 5 weeks each. New growth leaves were harvested once every week during each of these time periods. All samples were carefully prepared and analyzed to determine the relationship of the 11B/10B ratio in the leaves compared to that of the supplied nutrient solution. It was observed that the 11B/10B isotope ratio of the growth solution had an influence on the 11B/10B stable isotope ratio in new growth leaves of vine plants.

M.Sc. / The strontium isotope ratio 87Sr/86Sr in the wine and soil of four wine-producing regions in South Africa has been determined using quadrupole ICP-MS. Isotope ratio measurements with fairly good precision had been obtained and 87Sr/86Sr ratio values in wine and soil samples were statistically evaluated to determine whether the regions investigated could be distinguished. Statistical interpretation of data revealed that the Robertson wine-producing region could be distinguished from Stellenbosch, Swartland and Walker Bay wine regions on the basis of their 87Sr/86Sr ratio. By using a combination of 87Sr/86Sr and multielement data, canonical discriminant functions with very good classification abilities could be obtained to correctly distinguish between wine and soil samples originating from a specific production region. Due to the isobaric overlap of strontium and rubidium at mass 87, a very good strontium-rubidium separation method had to be developed. The chelating properties of EDTA and DCTA in combination with ion exchange chromatography were explored in order to achieve such a separation. Due to the advanced rubidium separation ability of the developed DCTA elution method, it has been employed in all wine and soil sample preparations. Since chromatographic ion exchange procedures are not capable of removing all the rubidium from the sample matrices, 87Sr/86Sr ratio values were mathematically corrected for residual rubidium using an 88Sr correction. Since strontium isotope ratio measurements require very good precision, it is traditionally determined using TIMS. However, due to the ease of operation, shorter analysis time and widespread availability of quadrupole ICP-MS, the technique was evaluated for its merit for determining strontium isotope ratios. The optimization of acquisition parameters for the determination of the 87Sr/86Sr ratio in wine and soil samples with good precision of below 0.1% RSD has been conducted. Furthermore, selected sample matrix components were assessed as possible matrix interferences as well as for their ability to affect the precision of measurements. It was concluded that with adequate optimization of acquisition parameters, proper sample preparation and reliable mathematical correction of data, 87Sr/86Sr ratio measurements with reasonable precision could be obtained using quadrupole ICP-MS.

M.Sc. / The South African wine industry is well respected internationally for producing high quality wines. The possible adulteration of these wines can lead to loss of reputation and a loss of sales and could also be dangerous to consumer’s health. Multi-element analysis of wines is one way of implementing quality control and the same multi-element data can also be used to prove the point of origin. The metal content of the fruit (grapes) should represent the metal content of the soil in which the plants (vineyards) were grown. An Inductively Coupled Plasma Mass Spectrometer (ICP-MS) was used with correct internal standard and interference correction to obtain reliable concentrations for 27 elements (Li, B, Al, Sc, V, Cr, Mn, Co, Ni, Cu, Zn, Se, Rb, Sr, Zr, Nb, Mo, Cd, Sn, Sb, Ba, Ce, Nd, W, Tl, Pb and U) of 1:1 diluted wines and microwave digested vineyard soil from four South-African wine-producing regions: Stellenbosch, Swartland, Robertson and Walker Bay. This multi-element data was then interpreted using multivariate statistical analysis in order to determine which elements have the ability to discriminate between the four regions. Li, B, Sc, Ni, Mn, Co, Cu, and Rb were the elements that were identified to have discrimination ability. 96% of wines and 100% of vineyard soils were correctly classified. Indirectly it has been proven that the metal content of the soil can be correlated to the metal content of the wine. This methodology can be reliably used in industry for quality control and routine provenance determination

A study was undertaken to compare growth potential of three Eucalyptus species of natural origin in South Eastern Australia, with commercially grown species on mid and high altitude temperate sites in the summer rainfall growing zones in South Africa. The three species were Eucalyptus globulus subsp. bicostata (E. bicostata), Eucalyptus cypellocarpa and Eucalyptus nobilis. Nine sites were selected to represent the growing areas of South Africa experiencing cold winter drought with occasional snow falls. Improved, commercially grown pure Eucalyptus species and interspecific hybrid clones were included as controls in the trials to give comparative growth performances on specific sites. Both balanced and unbalanced lattice designs were used to evaluate the growth potential of the unimproved Australian species and the improved commercial controls incorporated into the trials. Volume production and basal area growth were assessed for the three species at all sites. However, only three sub species E.globulus (E. bicostata, E. maidenii and E. globulus) showed varying levels of disease (Mychosphaerella nobilosa) infestation and this was therefore assessed in trials at 12 and 30 months respectively, and correlation analysis was used to study the relationship between the impact of disease infestation and growth performance. It was found that negative phenotypic correlations existed between the levels of infection and tree growth. ie greater infection slower growth. Evaluation of genotype x environment interaction (GXE) revealed that this did not exist in the E. bicostata nor E. nobilis populations, and therefore one population of each species can be developed for all the sites tested. E. cypellocarpa was the only species that showed some GXE interaction implying that separate populations to be developed for different sites. Contrary to what was expected, unimproved Eucalyptus species being investigated performed equally as well as the improved commercial species included as controls, thus providing potential for commercial deployment with selection and breeding. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.