A large sector of the forestry industry of South Africa comprises Eucalyptus species, covering approximately 49% of the forestry plantation area. Polyploidy induction has become an attractive tool to increase yield and reduce invasiveness in forestry species. Polyploidy induction in Eucalyptus using colchicine treatments on seed and axillary buds was undertaken to produce tetraploids that could be used in breeding programmes; specifically to increase yield and decrease species invasiveness through the production of triploids after crossing with diploid parents. Eight seedlots of E. urophylla and seven of E. grandis were treated with four colchicine concentrations (0.00, 0.01, 0.03, 0.05%) at two exposure times (18 h and 24 h), treating two seeds per treatment, repeated eight times. For axillary bud induction, 20 buds of two E. grandis clones and three E. grandis × E. urophylla hybrids and one E. grandis × E. nitens hybrid were treated with four colchicine concentrations (0.0, 0.5, 1.0, 1.5%) for three consecutive days. A known tetraploid hybrid E. grandis E. camaldulensis and its corresponding diploid were included as reference material. Seedlings and bud sports were pre-screened by determining stomatal guard cell lengths. Seedlings and bud sports displaying cell lengths significantly (p<0.0001) larger than the diploid were selected as putative polyploids. Polyploidy was then confirmed by quantifying the DNA content using flow cytometry. Stomatal frequencies and guard cell chloroplast frequencies were also determined in the induced tetraploid seedlings to evaluate their suitability to discern between ploids. All putative polyploidy seedlings, identified in the pre-screening process, were confirmed, using flow cytometry, as either tetraploids or mixoploids. Of the 17 E. urophylla putative polyploids, from various seedlots, six were tetraploid and 11 mixoploid. In E. grandis one of the five putative polyploids, from various seedlots, was tetraploid and four mixoploid. Pre-screening of bud sports was less accurate; only four of the 12 E. grandis hybrid putative polyploids were mixoploid and only three of the six E. grandis putative polyploids were mixoploid. E. urophylla seedlings were more sensitive to colchicine than E. grandis seedlings displaying a lower survival rate (52%) than E. grandis (63%). Extreme treatments that caused the lowest survival rates were also responsible for most of the polyploidy successful inductions; 0.05%/18 h and 0.05%/24 h for E. urophylla and 0.03%/24 h and 0.05%/24 h for E. grandis. Phenotypic effects of colchicine included shorter, thicker roots and hypocotyls; darker leaves; longer and narrower leaves in some tetraploids; and asymmetrical leaf margins in many mixoploids and tetraploids compared with the controls. In the tetraploids, stomata were significantly larger (p<0.0001) and less frequent (p<0.001). A significant (p<0.001) increase in the number stomatal chloroplasts was also ascertained. Confirmed mixoploid seedlings all displayed tetraploid leaves based on stomatal size and thus classified as periclinal chimeras. In bud sports, only leaves with islands of diploid and tetraploid stomata in the confirmed mixoploids were encountered. Mixoploid bud sports were thus either sectional or mericlinal chimeras. Stomatal size proved to be a suitable pre-screening method, especially in polyploidy induction in seedlings. Additionally confirmed tetraploids exhibited significantly different stomatal frequencies and stomatal chloroplast frequencies compared with the diploids, thus proving to be suitable detection methods for polyploidy screenings. Polyploidy induction in seed was effective, however, less effective in axillary buds which requires further research to refine methods. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

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.

The first objective of this study was to evaluate Eucalyptus urophylla S.T. Blake provenances in terms of their growth, basic wood density and pulp yield properties. The second objective was to determine the genetic and phenotypic associations that may exist between growth, basic wood density and pulp yield. Data of 9022 open-pollinated progenies representing 306 families, collected from 17 provenances, were used to evaluate growth. To evaluate basic wood density and pulp yield, as well as the genetic and phenotypic associations between the three traits, data of 300 open-pollinated progenies representing 30 selected families from 11 provenances were used. Narrow-sense heritabilities for all three traits were estimated from data collected in a single E. urophylla provenance/progeny trial planted in northern KwaZulu-Natal. The results showed that significant provenance effects for growth, basic wood density and pulp yield were observed. Heritability was found to be strong for basic wood density (h2 = 0.51) and moderate to weak for volume growth and pulp yield (h2 = 0.17 and h2 = 0.11, respectively). This suggests that big genetic gains can be achieved for basic wood density. Although the heritability estimates for volume growth and pulp yield were weaker, this still allows for tree breeders to make significant genetic gains through accurate selection from this E. urophylla breeding population. Genetic and phenotypic associations between the three traits were estimated from data collected in the same trial. The genetic correlation between volume growth and pulp yield was positive and moderately strong (rA = 0.66). The genetic correlation estimate between volume growth and basic wood density was found to be negative but weak (rA = -0.08). The genetic association between pulp yield and basic wood density was found to be positive but weak (rA = 0.17). Correlation estimates between volume growth and basic wood density, as well as between pulp yield and basic wood density produced standard errors greater than the correlation itself (s.e. = ± 0.32 and ± 0.22, respectively). These high standard errors, coupled with weak genetic correlations, suggest that these correlation estimates are non-significant, but are probably a result of utilizing a small sample size. However, these correlations have a value in making breeding choices, if treated with caution. Key words: Eucalyptus urophylla, provenance, growth, basic wood density, pulp yield, heritability, genetic correlation / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Clones of the Eucalyptus grandis x Eucayptus nitens (GN) hybrids were produced and selected through the CSIR‟s breeding programmes for colder plantation sites in South Africa. Some GN clones consistently exhibit high and superior pulp properties, which makes them valuable for commercial plantations in South Africa. In nurseries, stockplants are usually seven cm in length and maintained at high (100 x1.5 m-2) planting density. However, rooting frequency varies with season and little is known about the impact of position of cuttings on overall rooting frequency of a clone. The aim of this study was to investigate the effect of size and planting density of stockplants in mini-hedges, on the yield and subsequent rooting of cuttings from various positions of GN clones of known rooting potential (i.e. GN 018B: difficult-to-root and PP 2107: easy-to-root clones). Stockplants (10 cm vs. 20 cm) were established at high (100 x 1.5 m-2) and at low (25 x 1.5 m-2) densities for GN 018B and PP 2107 under commercial nursery conditions in a polyethylene tunnel. Cuttings were harvested every two to three weeks in September-October 2010 (spring), December 2010-January 2011 (summer), April-May 2011 (autumn) and June-July 2011 (winter). The harvested material was 5 – 7 cm in length and the light intensity received by individual stockplants at the two planting density levels was recorded. Harvested cuttings from the three positions (apical, middle and basal shoots) were used for: (i) rooting experiments under nursery conditions, (ii) bio-stimulant analysis using the mung bean bioassay, and (iii) analysis of soluble sugars. Between spring and summer 2010, the two GN clones established at low density yielded a similar number of cuttings, but differences in the rooting frequencies were significant in favour of PP 2107 clone. Similar observations were made at high density in terms of production of cuttings, but the significant differences in the rooting observations were reversed between the clones. The GN 018B clone had low rooting rates in summer under nursery conditions but its tissue extracts promoted higher rooting in the bioassay during that time, when compared to spring. Spring and summer had similar effects on rooting responses of PP 2107 cuttings in nursery and bioassay experiments. For both clones, short stockplants produced fewer cuttings but had a higher rooting frequency than cuttings from tall stockplants, with a high rooting frequency recorded from basal cuttings. Similar results were observed in the bioassay experiments which showed high rooting potential of mung bean hypocotyls cuttings using tissue extracts of PP 2107 cuttings maintained at high planting density. Although apical cutting tissues had high concentration of sugars (i.e. sucrose, glucose and fructose), their rooting rates were usually lower at high and low planting density compared to middle and basal cuttings. Sucrose concentration was the highest sugar present in stockplants grown under low planting density. A higher and lower rooting frequency was also observed in autumn although the two clones responded differently to Quambalaria eucalypti (Sporothrix eucalypti) disease infestations. Position, size and genotype had a significant impact on type and concentration of sugar (i.e. sucrose, glucose and fructose), particularly in PP 2107 clone, although rooting rates in the bioassay did not correlate with sugar contents of Eucalyptus cuttings. High carbohydrate (i.e. soluble sugar) content and auxin concentration increased production and subsequent rooting of cuttings across both clones, particularly in spring. Furthermore, rooting was enhanced by relatively higher light intensity intercepted by individual stockplants and in particular the GN 018B clone. Light intensity in the high and low planting densities caused variation in the rooting frequencies thereby increasing or decreasing soluble sugar and auxin concentrations of the two clones. Light intensity and fertiliser concentration received by tall and short stockplants impacted on endogenous hormone levels thereby increasing or decreasing rooting. High sugar concentration levels of PP 2107 clone increased its susceptibility to fungal infection thereby decreasing its rooting frequency in autumn, as its rooting rates increased in winter. Overall results of the investigation revealed that PP 2107 clone has higher rooting potential than GN 018B clone, in particular at high planting density and if stockplants are not infected by fungal diseases. Higher sugar levels were recorded in spring for PP 2107, although rooting rates of mung bean hypocotyl cuttings were higher in summer for GN 018B, suggesting that sugars have nothing to do with rooting of GN cuttings. Season, planting density and size of stockplants affect the rooting frequency of GN clone. Thus, short stockplants maintained at low and high planting densities are recommended for GN 018B and PP 2107 respectively, although the impacts of fertilisers and pathogen resistance on rooting rates still need to be investigated under similar conditions. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Eucalyptus macarthurii is one of the cold tolerant eucalypt species grown in South Africa for pulp and paper. However, little research has been done on this species’ growth performance. A study was therefore initiated to: i) analyse growth characteristics of Eucalyptus macarthurii at two sites and to calculate genetic parameters (genetic and phenotypic correlations, heritabilities and genetic gains), ii) develop a non-destructive near infrared calibration model to predict whole tree pulp yield of Eucalyptus macarthurii, and iii) screen a second generation Eucalyptus macarthurii breeding population, using the developed near infrared calibration model on core samples, to predict screened pulp yield and to rank and identify families with superior pulping properties. Eucalyptus macarthurii population growth data (diameter under bark, diameter over bark, bark thickness, bark stripping, height, basic wood density and stem form) were measured at Pinewoods and Vlakkloof sites and their respective genetic parameters calculated. Genotype by environment interaction was found in this population, indicating that different populations should perhaps be developed independently of each other for the two sites. Genetic and phenotypic correlations between diameter over bark and diameter under bark were, 0.96 and 0.98 for Pinewoods and 0.98 and 0.99 for Vlakkloof, respectively. These correlations indicated that selection of diameter over bark would lead to a positive indirect selection for diameter under bark. The heritability estimates also ranged from 0.03 to 0.23 at both sites, which indicated a reasonable response to selection. The predicted gains for all traits found at Pinewoods were higher than those at Vlakkloof for progeny trials E76/P1, except height for progeny trial E76/P2, which was 2.09m at Pinewoods site and 3.52m at Vlakkloof site which showed that, selection for taller trees will be more effective at Vlakkloof site. A preliminary study was undertaken from eleven second generation trees (2007 tree collection) to investigate if the radial strip core taken at breast height predicts the whole tree wood properties. Correlations found between laboratory Kraft pulping of whole tree wood discs and whole tree NIR spectra with that of the radial strip core NIR spectra were 0.9472 and 0.9506, respectively. These results confirmed that NIR spectra of the radial strip core at breast height predict the whole tree wood properties. A non-destructive near infrared calibration model using wood samples was obtained from Eucalyptus macarthurii felled trees. The wood samples were chipped into wood chips, pulped using Kraft pulping (reference method) and a sub-sample of wood chips of the same trees were ground into sawdust samples and analysed through near infrared spectroscopy for screened pulp yield. The screened pulp yield values obtained from both processes had a narrow screened pulp yield range of 40 to 48%. The Eucalyptus macarthurii screened pulp yield values obtained from both processes, as well as from values obtained from other eucalypt species, were subjected to Vision® Software for calibration and validation of the near infrared calibration model. The results indicated a strong calibration correlation coefficient of 94%, between Kraft pulping and near infrared spectroscopy with a validation coefficient of 89%. The strong correlation and validation coefficient indicated that a reliable non-destructive near infrared model to predict screened pulp yield was successfully developed. The successful development of the valid calibration model required a wider range of other eucalypts species, which improved the development of the model. The developed calibration model was applied to the second generation breeding population planted in KwaZulu-Natal and Mpumalanga provinces, using wood core samples obtained from standing trees for the prediction of screened pulp yield. The highest screened pulp yield achieved was 48%, which compared well to that found for Kraft pulping, which confirmed the success of the development of the calibration model. There was a wide scope of growth variation found amongst traits, which will be useful in selecting superior trees for the next generation. The development of the nondestructive near infrared calibration model was a success due to the strong correlation coefficients found between the screened pulp yields obtained from Kraft pulping and near infrared spectroscopy processes, which was achieved by the inclusion of other eucalypt species in the dataset. The calibration model can be used to select the top performing individual and family trees for the next generation based on screened pulp yield. Tree improvement trials can now be conserved for further breeding, without felling the trees for determination of pulping properties. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.