Please read the abstract in the 00front part of this document. / Dissertation (MSc Agric (Weed Science))--University of Pretoria, 2001. / Plant Production and Soil Science / Unrestricted

Thesis (MScFor (Forest and Wood Science))--University of Stellenbosch, 2007. / This study evaluates the variation that exists between six sites and between more than 200 Pinus patula families established across the sites for various traits utilised in a tree improvement programme. The traits utilised were growth at ages five and eight years, gravimetric densitometry of a sub-sample of the top 100 families at age eight, and microdensitometry and fibre morphological characteristics as determined by image analysis of increment cores, extracted from a sub-sample of the top 30 families. Significant differences were found between locations for growth at ages five and eight, density (both gravimetric and micro densitometry) and fibre properties. For each of the site combinations, utilising Type B- genetic correlations, the interaction between families and locations were evaluated in order to determine which locations could be grouped together in order to determine the effect various sites will have on the deployment of material. Significant differences were also found between families for the various traits investigated, which would indicate that desired trait or trait combinations can be selected for in a tree improvement programme. Heritability estimates for growth varied across sites, ranging from 0,32 to 0,57 at five years and 0,34 to 0,59 at eight years for family heritability. The individual tree estimates ranges from 0,08 to 0,27 at five years and from 0,09 to 0,26 at eight years. The standard errors associated with the heritability estimates for growth however indicate that the estimates, especially those of the individual trees should be used with caution. The heritability estimates for density and fibre morphological characteristics on the family and individual tree level are on a number of sites very high, although this is associated with large standard errors. Indications were that the traits can be combined effectively into a multi-trait selection index, since the phenotypic and genotypic correlations indicated mostly favorable or slight negative correlations between traits.

Thesis (MScFor (Dept. of Forest and Wood Science)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In this study 1345 pieces of 38x114 mm timber sawn from 16-20 year-old Pinus patula trees were obtained from a wide variety of sites along the Mpumalanga escarpment in South Africa. The samples were tested for various mechanical and physical properties. The objectives of this study were (1) to determine the variation in the bending strength, tension strength, and stiffness of the sawn timber, (2) to assess the efficiency of the current visual and mechanical grading rules on this sample, and (3) to evaluate the potential of some indicator properties to be used as structural grading parameters on this resource. A large number of non-destructive measurements were performed on all the samples, including scanning of the boards with a commercial X-ray density scanner, moisture content measurement, growth ring measurements, warp measurement, acoustic frequency measurement and measurement of the stiffness using a mechanical grader. The data from the X-ray density scanner was used to calculate a number of knot-related variables for each board. Visual grading according to the SANS 1783-2 (2005) specifications for structural timber was performed by a certified grader on all the boards. The timber was divided into two groups for destructive testing, one group each for bending and tension tests. Tests were conducted according to the SANS 6122 (1994) method for in-grade testing. Due to the sampling method followed, the destructive tests effectively used a random placement of defects with regard to the load application position. From the destructive tests the modulus of elasticity (MOEedge), bending strength (MOR) and tensile strength were determined. The study showed that the MOEedge of the sawn timber was far below the requirements of the current national standard (SANS 10163-1) for all of the visual and mechanical grades. The mean MOEedge of the visually graded S5 timber was about 30% lower than required. The 5th percentile values for bending strength of all the visual and mechanical grades were above the required values of SANS 10163-1. The 5th percentile values for tensile strength of all the visual and mechanical grades were similar to that required by the SANS 10163-1 standard. Correlations between flatwise measured MOE (MOEflat) and edgewise measured MOE (MOEedge) were smaller than expected, as well as the correlations between both MOEedge and MOEflat with MOR. Dynamic MOE (MOEdyn), calculated from acoustic frequency tests on the timber, was found to be the best single predictor of MOEedge, MOR and tension strength. Multiple regression analysis showed that a combination of MOEdyn, density and knot parameters can be used to improve the predictability of some of the strength and stiffness characteristics of the timber. It is recommended that a comprehensive study on the structural grading of SA Pine be performed which includes (1) an analysis of market requirements in terms of strength and stiffness properties of timber, (2) in-grade testing of a representative sample of structural timber in South Africa, and (3) a review of the standards used in South Africa to regulate the structural grading of timber. / AFRIKAANSE OPSOMMING: In hierdie studie is 1345 stukke 38x114 mm 16-20 jaar-oue Pinus patula planke, afkomstig van ‘n wye verskydenheid groeiplekke teen die Mpumalanga platorand in Suid Afrika, gebruik. Die planke is getoets vir verskeie meganiese sowel as fisiese eienskappe. Die doelwitte van hierdie studie was om (1) die variasie in buigsterkte, treksterkte en styfheid van die gesaagde planke te bepaal, (2) die effektiwiteit van die huidige visuele -en meganiese graderingsreëls op hierdie monster planke vas te stel, en (3) die potensiaal te evalueer van sommige eienskappe wat gebruik kan word as strukturele graderingsparameters. ‘n Groot hoeveelheid nie-destruktiewe toetse is op alle planke uitgevoer, wat ingesluit het skandering van planke met ‘n kommersiële X-straaldigt-heidskandeerder, metings van voginhoud, groeiringwydtes, deformasie, akoestiese frekwensie en die bepaling van styfheid met behulp van ‘n meganiese gradeerder. Die data van die X-straalskandeerder is gebruik om ‘n aantal kwasverwante veranderlikes vir elke plank te bereken. Visuele gradering is op alle planke uitgevoer ooreenkomstig met die SANS 1783-2 (2005) spesifikasies vir strukturele hout deur ‘n gesertifiseerde gradeerder. Die hout is in twee groepe opgedeel vir destruktiewe toetse, een vir buigtoetse en een vir trektoetse, ooreenkomstig met die SANS 6122 (1994) metode vir binnegraadse toetse. As gevolg van die monsternemingsmetodiek wat gevolg is, is daar effektief gebruik gemaak van ‘n lukrake plasing van defekte met betrekking tot die las-aanwendingsposisie. Die modulus van elastisiteit (MOEedge), buigsterkte (MOR) en treksterkte is deur middel van destruktiewe toetsresultate bepaal. Die studie het aangetoon dat die MOEedge van gesaagde hout aansienlik minder as die vereiste van die huidige nasionale standaard (SANS 10163-1) is vir al die visuele en meganiese grade. Die gemiddelde MOEedge van die visueel-gegradeerde S5 planke was omtrent 30% laer as vereis. Die 5de persentiel waardes vir buigsterkte van alle visuele en meganiese grade was hoër as die vereiste waardes soos voorgeskryf deur SANS 10163-1. Die 5de persentiel waardes vir treksterkte van alle visuele en meganiese grade was gelykstaande aan wat vereis word deur SANS 10163-1. Korrelasies tussen MOE, gemeet op die wydte sy (MOEflat), en MOE, gemeet op die diktesy (MOEedge), asook die korrelasies van beide MOEedge en MOEflat met MOR van die planke was laer as verwag. Dinamiese MOE (MOEdyn), wat bereken was vanaf die akoestiese frekwensie resultate, is vasgestel as die beste enkele indikator van MOEedge, MOR en treksterkte. Meervuldige regressie analise het aangetoon dat ‘n kombinasie van MOEdyn, digtheid en kwasparameters gebruik kan word om die voorspelbaarheid van sommige van die sterkte- en styfheids eienskappe van die hout te verbeter. Daar word aanbeveel dat ‘n omvattende studie gedoen word op die strukturele gradering van SA Dennehout, wat insluit (1) ‘n analise van die markbehoeftes in terme van die sterkte- en styfheids eienskappe van hout, (2) binnegraadse toetsing van ‘n verteenwoordigende monster strukturele hout in Suid Afrika, en (3) die hersiening van standaarde in gebruik in Suid Afrika om die strukturele gradering van hout te reguleer.

Master of Science - Science / The replacement of indigenous grasslands by commercial forestry plantations on the mountainous eastern escarpment of South Africa, has inevitably led to alteration of nutrient cycling. The overall objective of this study was to determine the nutrient status (soil, litter and foliar nutrient concentrations) of Pinus patula and Eucalyptus grandis plantations and adjacent grasslands. The 2, 13 and 30 year old P. patula plantations and adjacent Driekop grassland were located relatively high up on the escarpment (altitudes ranging between 950–1610 m) and were underlain by dolomite while the 2, 8 and 13 year old E. grandis plantations and adjacent Brooklands grassland were located lower down (altitudes ranging between 900–1100 m) and were underlain by granite. It was assumed that the nutrient status of the soil under the plantations and the adjacent grasslands were similar prior to afforestation. Samples were collected once in winter (August 2002) and once in summer (January 2003). Soil samples were collected to a depth of 20 cm and litter material, to a maximum depth of 20cm. Lower canopy foliar samples were collected within the lowermost metre of the canopies and upper canopy foliar samples were collected within the uppermost metre of the canopies. The data were analysed in the laboratory for various elements and soil physical parameters. The soil pH was lower under the P. patula (pH 4.8-5.2) and E. grandis plantations (pH 4.6-5.3) when compared with the soil under the adjacent grasslands (pH 5.3-5.6). The soil under the P. patula plantations had lower exchangeable base cation concentrations (204-300 mg kg-1) compared with the adjacent Driekop grassland (452-645 mg kg-1), while there were no clear trends when comparing the exchangeable basic cation concentrations in the E. grandis plantations and the adjacent Brooklands grassland. The soil aluminium saturation was similar between the plantations, ranging between 39.7 and 63.3% in the P. patula plantations and 27.4 and 75.6% in the E. grandis plantations. The grasslands had lower soil aluminium saturations, ranging between 17.7 and 35.7% in the Driekop grassland and 17.5 and 39.1% in the Brooklands grassland. Exudates from mycorrhizal associations chelate iv acidic cations, rendering plantations more tolerant of acidic soils. Soil total nitrogen, phosphorus and carbon in the plantations were similar to the adjacent grasslands. Higher nitrogen mineralisation rates in the P. patula plantations corresponded with higher litter and foliar total N concentrations relative to the adjacent Driekop grassland, while there were no clear trends when comparing rates of N mineralisation in the E. grandis plantations and the Brooklands grassland. The concentrations of foliar amino acids and protein were significantly higher (p<0.01) in the plantation foliage, when compared the grassland foliage. The amino acid arginine, occurred in higher proportions relative to other amino acids in plantation foliage, when compared with the relative proportions found in the grasslands. Relative to nitrogen, foliar ratios indicated that phosphorus and potassium were the most limiting nutrients for the plantations, which possibly resulted in N storage in the form of protein and amino acids. The soil and foliage were the more sensitive indicators of the nutrients status than litter. Differences in nutrients concentrations between the lower and upper canopies in both plantations were minor and no major trends were found. Therefore the sub division of lower and upper canopies is not recommended for further foliar nutrient analyses. Age related trends were unclear as a result of higher soil clay contents found in the 13 year old P. patula plantation and the 8 year old E. grandis plantation as well as the various silvicultural practices implemented. In terms of the relative impacts of P. patula and E. grandis plantations, the results of this study imply that the upper 20cm of soil was more impacted under the P. patula plantations than under the E. grandis plantations, in terms of reduced base cation and increased acid cation concentrations. The P. patula plantations are shallow rooters and thus the base cation status of the upper soil horizons may affect the sustainability of the P. patula plantations.

Embryogenic tissue of Pinus patula Scheide et Deppe was initiated from immature green female cones during the months of November 1996 to February 1997 and December 1997 to January 1998. Tissue was maintained on MSG3 medium (BECWAR, NAGMANI and WANN 1990) supplemented with maltose. A comparison of various sugars as a carbohydrate source for maintaining embryogenic tissue showed that maltose was far superior to sucrose and the other sugars tested. Embryogenic tissue was successfully cryopreserved for up to 8 weeks using 0.3 M sorbitol and 5 % DMSO. Recovered tissue initially underwent a lag phase in tissue regrowth, but by the end of 5 weeks post-thaw, tissue proliferation was as vigorous as the unfrozen, untreated control. Fluoresceine diacetate (FDA) staining revealed that the embryonal head survived cryopreservation, but the highly vacuolated suspensor tissue had ruptured and died. Embryogenic tissue from two different families and four genotypes were successfully cryopreserved using this protocol. A comparison of commonly used cryopreservation techniques was conducted. It was found that the slow addition of the cryoprotectants over two days slowed the recovery rate of the tissue and increased the chances of contamination. Embryogenic tissue did not respond well to cryopreservation using a combination of the cryoprotectants PEG, glucose and DMSO (10-8-10%). Only a small proportion of the tissue survived, and initial tissue regrowth took up to 5 weeks. Embryogenic tissue was also set in gel and immersed directly in liquid nitrogen in an effort to cryopreserve tissue using the process of vitrification. However, none of the tissue survived, possibly due to insufficient dehydration prior to immersion in liquid nitrogen. Tissue recovery was highest when the tissue was precooled to -70°C in a container filled with isopropyl alcohol placed in a static freezer prior to immersion in liquid nitrogen. Recovery of tissue was improved by suspending the tissue on polyester grids and removing the liquid medium prior to placing onto MSG3 medium. Recovered tissue was bulked up using suspension cultures, and then paced onto MSG5 (BECWAR, NAGMANI and WANN 1990) or 240 medium (PULLMAN and WEBB 1994) to mature. Mature embryos were isolated from both media and germinated. Somatic plantlets were successfully hardened-off under greenhouse conditions. The successful cryopreservation of a number of genotypes and lines, and the maturation of recovered tissue has been achieved. This technique is now being actively incorporated into P. patula somatic embryo research, enabling the long-term storage of juvenile reference tissue while field trials are carried out and evaluated. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

Thesis (Ph.D. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2008.

Thesis (Doctor of Phil.) - University of KwaZulu-Natal, Pietermaritzburg, 2008. / Full text also available online. Scroll down for electronic link.

The objectives of this study were to study wood property variation in Pinus patula from different physiographic sites, the interrelationship between wood properties and the effects of some forest site factors on wood properties and their within-tree variation. The wood properties considered were density, transverse shrinkage, grain angle and stiffness (modulus of elasticity). Ten trees were taken from each of seventeen sites selected during a previous study of soil ecology and tree growth patterns on the escarpment of Mpumalanga in South Africa. This is a complex physiographic region with several different growth sites within a relatively small geographical area. The key finding of the study was that the effect of radial distance from the pith (i.e. the age of the tree) was highly significant and accounted for most of the variation of the trees sampled. Although the effect of site on the selected wood properties was highly significant, its effect accounted for a relatively small percentage of the total variation in each case. Consequently, solid wood processors must take into account the large variation within a tree when implementing strategies to minimise the downgrade of timber as result of excessive board distortion and/or inferior strength. There were significant correlations between wood density and density slope, radial shrinkage, shrinkage ratio and stiffness and also significant correlations between wood stiffness and density slope and radial shrinkage. Density and grain angle both showed significant correlations with the most important climatic factors. ‘Growth days’ (which is an index reflecting the number of days per year when soil moisture is not a limiting factor for plant growth) was identified as one of the principal growth factors influencing the selected wood properties, because of it’s strong relationships with wood density, density slope, grain angle and stiffness. Regression models were developed to predict wood density and grain angle from growth variables. The results show that those wood properties that correlate with geographic representable site variables can be included in site classification and evaluation systems, to assist in predicting growth in terms of volume yield and wood quality.

Pinus patula is the single most important commercial plantation forest tree species in South Africa. It accounts for 52% approximately (700,000 ha) of total commercial plantation area in the country and is utilised for saw logs and pulp and paper production. P. patula is a tropical pine species indigenous to Mexico. Excellent ex situ conservation and range-wide provenance trials have been established for P. patula in South Africa and South America. These highly organised trials provide the opportunity to perform association genetic studies with the long term aim to identify trait linked markers for future molecular improvement of P. patula. In this study, the first gene-based assessment of allelic diversity in P. patula was performed. This pilot study focused on two cellulose biosynthetic genes as representatives of wood formation genes and assayed molecular evolution parameters such as nucleotide diversity, allelic diversity and linkage disequilibrium (LD) in a species-wide reference population of P. patula. Two novel cellulose synthase (CesA) genes were isolated and characterised in P. patula. One of these genes, PpCesA1, is putatively involved in the biosynthesis of secondary cell walls of tissues such as xylem (wood), while the other, PpCesA2 is proposed to be associated with primary cell wall formation in rapidly growing tissue types. The genomic DNA copies of PpCesA1 and PpCesA2 were 6025 bp and 6365 bp in length, respectively. The corresponding cDNA sequences encoded 1083 and 1058 amino acids, respectively, and differed considerably from each other (73% amino acid identity). Both amino acid sequences contained the key domains and motifs characteristic of functional CESA proteins isolated in other higher plants. Phylogenetic analysis revealed that PpCesA1 was most similar (99%) to its putative ortholog in Pinus taeda, PtCesA3, and PpCesA2 was highly similar to a putative ortholog in Pinus radiata, PrCesA2 (99% identity). This phylogenetic analysis supported previous findings that the divergence between the primary and secondary cell wall associated CESA proteins occurred before the divergence of angiosperms and gymnosperms approximately 300 million years ago. A fragment of a putative paralogous gene copy of PpCesA1, named PpCesA1-B was also isolated. The PpCesA1-B gene fragment was found to differ from PpCesA1 by 22 nucleotide polymorphisms and its non-allelic (paralogous) status was confirmed by segregation analysis in P. patula. In order to gain an understanding of molecular genetic variation that might affect wood formation in P. patula, we sequenced multiple allelic variants of PpCesA1, PpCesA1-B and PpCesA2, which we sampled from a species-wide reference population of P. patula. The average levels of nucleotide diversity were found to be low for all three genes (π ≈ 0.0015), which may be a property of functional members of the CesA gene family. As a result of the low nucleotide diversity, only small numbers of pair-wise informative sites were available for LD analysis and the decay in LD could only be studied in PpCesA2 where it was found to decay very rapidly (within 200 bp). Tests of neutrality suggested that the exon sequences of PpCesA1 and PpCesA2 were under significant positive (adaptive) selection. Comparison of levels of nucleotide diversity and selection in different parts of the two genes indicated that the highest levels of adaptive selection occurred in areas where amino acid substitutions could alter protein structure or function. This study provides valuable insights for designing future allele discovery efforts in P. patula with the ultimate goal of developing gene-based markers for the molecular improvement of wood formation in this tree species. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

Progress in tree biotechnology initially trailed behind agricultural crops due to their long life cycle, difficult tissue culture and regeneration protocols, and their abundance in natural forests. However, rapid global deforestation rates, together with an increased world demand for pulp, paper and timber products, have prompted scientific and commercial focus to improve genetic timber stocks. South Africa, a tree-poor country (where indigenous forests are protected), has relied almost solely on exotic plantations to meet its demand for timber. A pioneer study investigating the feasibility of using direct (biolistic) and indirect (Agrobacterium-mediated) methods for gene transfer was undertaken in Pinus patula Schiede et Deppe, a Mexican softwood and a forerunner for saw timber, pulpwood and paper in the South African forest industries. The aim of the transformation methods was to impart herbicide resistance to the trees. This was achieved via the introduction of a bar-GUS pAHC25 cassette under the control of the ubiquitin promoter. To provide target material for transformation, two in vitro micropropagation pathways were used: somatic embryogenesis and organogenesis. Both embryonal suspensor masses (ESM) and somatic embryos at various stages of development were initially used as target explants for the biolistic study using an established in vitro protocol. A stepwise selection was implemented in order to allow transformed (particularly bombarded) cultures the opportunity to regenerate under selection pressure using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l ¯¹ followed by 3 mg l¯¹ active ingredient at the next subculture. Biolistic transgene delivery was more efficient when sorbitol was included in the pre-bombardment medium enabling use of higher vacuum and shooting pressures, without lowering the regeneration potential of ESM significantly. Bombarded material from two genotypes (Lines 2 and 3) was regenerated to produce mature somatic embryos using an optimized regeneration regimen. The indirect study with Agrobacterium tumefaciens (LBA4404), transformed with the pAHC25 vector via triparental mating or heat shock, used a variety of target tissues including: mature somatic embryos, ESM and mature zygotic embryos (MZE's) - a novel in vitro system for P. patula. The Agrobacterium-mediated method resulted in optimized decontamination conditions using a combination of liquid MSG3 (or sterile dH₂O for mature embryos) supplemented with 500 mg l ¯¹ cefotaxime, with rotation, and sterile 65 mm Whatman No. 3 filter paper stacks, which avoided excess filtering and stress to transformation material. Further efforts to aid regeneration during the indirect study included L-proline post-transformation, though no mature somatic embryos were regenerated at the conclusion of the Agrobacterium-mediated study. Recovery of transformed ESM in both studies was best during the active growth phase 4-6 d after subculture. Regeneration with good somatic embryo potential was an exigent aspect in both transformation studies. Expression of positive histochemical GUS activity in all transformed material was confirmed by polymerase chain reaction (PCR) analysis indicating that Pinus patula tissue was amenable to transformation. A new bar PCR regime was implemented in P. patula. In the biolistic study, a higher transformation efficiency of bar amplicons (53%) than GUS amplicons (45%) was observed, reflecting their non-linked status on the pAHC25 transformation vector. This is the first report of biolistic transformation of P. patula that will allow for the production of transgenic ESM. The production of transgenic P. patula holds great promise for commercial development in the South African forestry industry. The application of transgenic trees in the timber industry is numerous but the aims most relevant to P. patula include wood modification and disease resistance to pathogens like pitch canker fungus. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.