Eucalyptus trees are an important source of wood and fibre. The wood (secondary xylem) of this genus is widely used for pulp and papermaking. However, our understanding of the mechanisms which control the wood formation process (xylogenesis) in Eucalyptus and other woody species is far from complete. One reason is that xylogenesis is a very complex developmental process. The major components of wood are lignin and cellulose. Many genes involved in lignin and cellulose biosynthesis have been characterized. For example, Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two important lignin biosynthesis genes. Plant cellulose is synthesized by cellulose synthase enzymes with the aid of some other proteins, such as sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Another factor which makes it difficult to analyze the function of Eucalyptus wood formation genes in vivo, is the long generation times of Eucalyptus trees and the difficulty to obtain transgenic Eucalyptus plants. Therefore, in this study, we investigated the use of Arabidopsis thaliana as a model system for functional analysis of wood formation genes. We transformed a lignin and a cellulose biosynthesis gene isolated from Eucalyptus to wild-type and mutant genetic backgrounds of Arabidopsis in order to test our ability to modify the cell wall chemistry of Arabidopsis thaliana using tree genes. The Eucalyptus CCR (EUCCR) gene was transformed into wild-type Arabidopsis (Col-0) and irregular xylem 4 (irx4) mutant plants, in which the homolog of EUCCR is mutated. A Eucalyptus cellulose synthase gene (EgCesA1) was also transformed into irregular xylem 1 (irx1) mutant plants, in which the homolog of EgCesA1 is mutated. Transgenics were only obtained from wild-type Col-0 transformed with EUCCR and from irx1 transformed with EgCesA1. We studied the cell wall chemistry of wild-type Arabidopsis plants overexpressing the Eucalyptus CCR gene. Chemical analysis of inflorescence stems revealed the modification of lignin and cellulose content in transgenic plants. Total lignin content was increased in T2 (5%) and T3 (12%) lines as revealed by micro-Klason lignin and thioglycolic acid quantification methods, respectively. High Pressure Liquid Chromatography (HPLC) analysis revealed that cellulose content was significantly decreased (10%) in T2 transgenic plants. This suggested the reallocation of carbon from cellulose to lignin as a result of overexpression of EUCCR in transgenic plants. Interestingly, thioacidolysis analysis revealed that in T2 plants, monomethoxylated guaiacyl (G) monomer was increased (16%) and bimethoxylated syringyl (S) monomer was decreased (21%). Therefore, the S/G lignin monomer ratio was significant decreased (32%). This implied that EUCCR might be specific to G monomer biosynthesis. The results described above confirmed that Arabidopsis thaliana can be used to model the function of wood formation genes isolated from Eucalyptus. Two novel full-length Eucalyptus sucrose synthase (SuSy) genes, EgSuSy1 and EgSuSy3, and one putative pseudogene, EgSuSy2, were also isolated in this study. Degenerate PCR was used to amplify Eucalyptus SuSy fragments from cDNA and genomic DNA. 3’RACE was used to amplify the 3’ ends of two Eucalyptus SuSy genes. Genome walking was performed to obtain the 5’ ends of EgSuSy1 and EgSuSy2 whereas 5’RACE technology was used to isolate the 5’ end of EgSuSy3. However, 3’RACE analysis failed when we tried to identify the 3’ end of EgSuSy2. Sequencing results from the genome walking product of EgSuSy2 further revealed that the start codon of this gene was missing, and we hypothesize that this is a psuedogene in the Eucalyptus genome. The EgSuSy1 cDNA was 2498 bp in length with an open reading frame of 2418 bp encoding 805 amino acids with a predicted molecular mass of 92.3 kDa. The 2528 bp full-length EgSuSy3 cDNA contained the same length of open reading frame as EgSuSy1, but encoded a polypeptide with a predicted molecular mass of 92.8 kDa. The results of quantitative real-time RT-PCR, phylogenetic analysis and gene structure of the two genes revealed that both genes might be involved in cellulose biosynthesis in primary and secondary cell walls of Eucalyptus. These two genes, EgSuSy1 and EgSuSy3, could therefore be useful targets for genetic engineering of wood properties in Eucalyptus. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Sustainable agriculture is a crucial factor to be considered in order to meet the growing demand for food production. The need for low cost and highly functional materials to provide the most efficient cultivation process has led the agriculture industry to consume petrochemical and mineral based material in an enormous amount. Thus, disposal of the used mulch materials has become a serious environmental issue. In this work, the possibility of using wood pulp fibre in two distinct applications in agriculture is investigated. First, agricultural mulching is the subject of the study and second, we focus on using wood pulp fibre as growing medium in greenhouses. Mulching in agriculture is an essential practice in order to have high crop yield, healthy products, and more efficient cultivation process. Over the years, agricultural mulch has been made out of a variety of materials. The most common of all is plastic mulch due to its low price and high functionality. However, the problems associated with applying and removing the enormous load of plastic and their disposal have made it an option far from ideal. Therefore, there is a need to develop mulches based on biodegradable materials. Paper-based mulch is one of the candidates, In the first chapter of this work, with a review of previous works in this area, we attempt to develop a new spray-able mulch based on wood pulp fibre. A novel foam forming method is utilised to deposit wood pulp fibres in combination with other chemicals as an evenly distributed fibre network on a porous bed. Currently available paper based-mulch is of a very high basis weight. In first part of this work, application of a foam formed low basis weight paper-based mulch is investigated. Whereas, in the second chapter, the use of wood pulp fibres in a similar function as “rockwool” in soilless greenhouse farming is investigated. Rockwool is named after fibres made of melted minerals at temperatures as high as 2000°C. Rockwool is used as blocks for seeds growth and propagation and as an alternative for soil in greenhouses. The feasibility of microenvironment control of the rockwool blocks in crop production plus its low cost have made is popular. However, their disposal has always been an environmental issue. The biodegradability of wood pulp fibres is a great advantage over mineral fibres used in rockwool. In the second chapter of current work, we study the possibility of using wood pulp fibres as carriers for agriculturally beneficial chemicals. Specifically, we focus on binding and release properties of small organic molecules from wood pulp fibres. The goal is to achieve an understanding of the capability of wood pulp fibres to be used in building biodegradable growing medium blocks in greenhouses. / Thesis / Master of Applied Science (MASc)