Increasing the sucrose yield and the disease resistance of plants are two major objectives of the transgenic sugarcane plant programme in South Africa. The sugarcane culm has thus been identified as one of the main target areas for transgene expression. A shortage of reliable promoter elements as well as patent limitations have necessitated the isolation of promoters that are preferentially expressed in the sugarcane culm. In the present study two different approaches were followed to isolate such promoters, and the bacterial promoter, rolC, was evaluated for tissue-specific expression in sugarcane. Differential display is a non-directed technique that was used to identify genes that are differentially expressed in the mature sugarcane culm. The original method was modified, and four putative culm-preferential fragments were isolated. Sequence and hybridisation analyses revealed that these fragments were false positives, and could therefore not be used to obtain a culm-specific promoter. Activity of the Agrobacterium rolC promoter was evaluated by analysing expression patterns of two reporter genes in the mature culm of transgenic sugarcane plants. Nucleic acid analyses indicated that the foreign DNA was incorporated into the sugarcane genome, and that mRNA transcripts were produced. Histochemical analysis was done to visualise rolC-driven GUS and GFP expression in the mature sugarcane culm. In both cases the reporter gene expression was restricted to the vascular bundles and specifically to the phloem. A directed approach was followed to isolate the gene and subsequently the promoter of the β-subunit of pyrophosphate-dependent phosphofructokinase (PFP-β). An incomplete cDNA clone was obtained from a mature culm cDNA library, and was used for the screening of a sugarcane genomic library. Two clones containing different parts of the PFP-β gene were isolated. A Deletion Factory™ system was used to analyse the clone containing the 5' end of the gene. The first five exons and 1747 bp of the 5' flanking region of the gene were sequenced. Preliminary activity analysis of the promoter region was done by constructing two expression vectors, and analysing transient GUS expression in sugarcane callus. Results indicated that the promoter is capable of driving foreign gene expression in callus. Transient expression levels were lower than that of the maize Ubi-1 promoter. Further analysis of the 5' flanking region will be done to establish whether cis-acting elements outside the analysed area have an influence on the activity of the promoter. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

The main project was an assessment of the effect that colonization by five different Vesicular-arbuscular mycorrhiza (VAM) cultures have on the growth of the indigenous-grasses Themeda triandra and Trachypogon spicatus, when grown on coalmine topsoil. With unamended topsoil, VAM showed the ability to significantly increase the growth of the grasses compared to non-VAM control plants. The amount of effect varied with the VAM inoculum culture type, with a VAM culture originally from the Cape Flats being the most effective. In a second trial, soil fertilized with nitrogen, potassium and low concentrations of phosphate (P) was used. Again VAM displayed the ability to improve grass plant growth. The increase in P caused the Large spore inoculum to become the most effective. This indicated that different VAM cultures are inhibited to different degrees by an increase in phosphate fertilization. The low level of VAM infection, in both trials, seemed to preclude most of the VAM associated nutrient uptake control. Varying reports have been published on the effect of fertilization on VAM infection and colonization. In an attempt to further elucidate the role of fertilizer in VAM inhibition, rhizosphere soil from a long term fertility trial near Witbank, S.A. was sampled. Amcoal environmental services fertilized forty-two plots with varying concentrations of nitrogen, potassium, phosphate and lime to assess the growth of a variety of grasses. The trial had been maintained for ten years before sampling was completed for this project. Samples from each plot were taken from the rhizosphere soil of the most prominent grass (Digitaria eriantha). VAM spores were extracted from all the samples and five different types of spores were identified and counted for each sample. By comparing spore counts from each plot, the effect that the fertilizer regime had on the VAM on that plot could be assessed. Variation in the concentrations of nitrogen (N) and potassium had no significant effect on VAM colonization. Very low concentrations of N could not be assessed as all plots had been initially top dressed with nitrogen fertilizer. Phosphate (P) fertilizer concentration had a marked effect on spore concentrations. There was a significant increase in spore concentration as P levels were increased from zero P fertilization to 80kgs P/ha. Further increase in P to ≥ 60kgs P/ha resulted in a significant decrease in spore concentrations. From this it would appear that a low level of soil P is needed to give maximum VAM colonization and further increase in soil P causes VAM inhibition. Lime ameliorated the VAM inhibition caused by high concentrations of P. Increase in P caused spore concentrations of low abundance propagules (LAP) too decreased more rapidly than high abundance propagules (HAP). In high P soils VAM with LAP would eventually be eliminated from the system resulting in a decrease in VAM diversity. A project was attempted to use the recently developed Randomly Amplified Polymorphic DNA in conjunction with the Polymerase Chain Reaction (RAPD PCR) techniques to identify different VAM families. The technique causes the amplification of segments of DNA which can be visualized by gel electrophoresis and staining. Band patterns formed can be related to the VAM of origin and hence used in identification of that VAM. An attempt was made to amplify DNA from a single spore in this manner which would, in conjunction with morphological observations, make identification of VAM easier and more accurate. Problems with either releasing the DNA from the spores, or substances in the spore inhibiting the PCR reaction made obtaining band patterns difficult. After many PCR attempts, varying extraction methods and PCR conditions, no repeatable results could be obtained and work on this project was discontinued. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

(Os gêneros Eriochrysis P. Beauv., Imperata Cirillo e Saccharum L. (Poaceae - Andropogoneae - Saccharinae) no Rio Grande do Sul, Brasil). A subtribo Saccharinae pertence à tribo Andropogoneae e inclui cerca de 140 espécies distribuídas em 13 gêneros, entre eles Eriochrysis, Imperata e Saccharum. O presente trabalho consiste em um levantamento das espécies desses três gêneros no estado do Rio Grande do Sul. Foram realizadas coletas intensivas em diferentes regiões fisiográficas do estado, bem como revisão de diversos herbários. Foi confirmada a ocorrência de três espécies nativas de cada um dos gêneros, no Rio Grande do Sul, além da “canade- açúcar” (Saccharum officinarum L.), cultivada no estado. A ocorrência dessas espécies no Rio Grande do Sul, embora já citada em trabalhos anteriores, constitui um acréscimo ao Catálogo de Plantas e Fungos do Brasil, recentemente publicado. A partir de materiais adicionais analisados, Eriochrysis villosa Swallen está sendo citada pela primeira vez para o estado do Paraná. São fornecidas chaves de identificação para as espécies confirmadas, descrições, informações sobre a sua distribuição geográfica, habitat e períodos de florescimento e frutificação, além de ilustrações de caracteres de importância taxonômica. / (The genera Eriochrysis P. Beauv., Imperata Cirillo and Saccharum L. (Poaceae - Andropogoneae - Saccharinae) in Rio Grande do Sul State, Brazil). The subtribe Saccharinae belongs to the tribe Andropogoneae and includes about 140 species, in 13 genera, including Eriochrysis, Imperata and Saccharum. This work consists of a survey of the species of these three genera in Rio Grande do Sul. Intensive field collections were made in different physiographic regions of the State, as well as revision of several herbaria. The occurrence of three native species of each genus was confirmed, in addition to the "sugar cane" (Saccharum officinarum L.), that is cultivated in the area. The occurrence of these species in Rio Grande do Sul, although mentioned in previous studies, should be added to the recently published Catalogue of Plants and Fungi of Brazil. Based on additional material examined, Eriochrysis villosa Swallen is being reported for the first time to Paraná State, Brazil. Analytical keys for the identification of the confirmed species, descriptions, data on their geographical distribution, habitat and periods of flowering and fruiting, as well as illustrations of important taxonomic characters, are provided.

(Os gêneros Eriochrysis P. Beauv., Imperata Cirillo e Saccharum L. (Poaceae - Andropogoneae - Saccharinae) no Rio Grande do Sul, Brasil). A subtribo Saccharinae pertence à tribo Andropogoneae e inclui cerca de 140 espécies distribuídas em 13 gêneros, entre eles Eriochrysis, Imperata e Saccharum. O presente trabalho consiste em um levantamento das espécies desses três gêneros no estado do Rio Grande do Sul. Foram realizadas coletas intensivas em diferentes regiões fisiográficas do estado, bem como revisão de diversos herbários. Foi confirmada a ocorrência de três espécies nativas de cada um dos gêneros, no Rio Grande do Sul, além da “canade- açúcar” (Saccharum officinarum L.), cultivada no estado. A ocorrência dessas espécies no Rio Grande do Sul, embora já citada em trabalhos anteriores, constitui um acréscimo ao Catálogo de Plantas e Fungos do Brasil, recentemente publicado. A partir de materiais adicionais analisados, Eriochrysis villosa Swallen está sendo citada pela primeira vez para o estado do Paraná. São fornecidas chaves de identificação para as espécies confirmadas, descrições, informações sobre a sua distribuição geográfica, habitat e períodos de florescimento e frutificação, além de ilustrações de caracteres de importância taxonômica. / (The genera Eriochrysis P. Beauv., Imperata Cirillo and Saccharum L. (Poaceae - Andropogoneae - Saccharinae) in Rio Grande do Sul State, Brazil). The subtribe Saccharinae belongs to the tribe Andropogoneae and includes about 140 species, in 13 genera, including Eriochrysis, Imperata and Saccharum. This work consists of a survey of the species of these three genera in Rio Grande do Sul. Intensive field collections were made in different physiographic regions of the State, as well as revision of several herbaria. The occurrence of three native species of each genus was confirmed, in addition to the "sugar cane" (Saccharum officinarum L.), that is cultivated in the area. The occurrence of these species in Rio Grande do Sul, although mentioned in previous studies, should be added to the recently published Catalogue of Plants and Fungi of Brazil. Based on additional material examined, Eriochrysis villosa Swallen is being reported for the first time to Paraná State, Brazil. Analytical keys for the identification of the confirmed species, descriptions, data on their geographical distribution, habitat and periods of flowering and fruiting, as well as illustrations of important taxonomic characters, are provided.

(Os gêneros Eriochrysis P. Beauv., Imperata Cirillo e Saccharum L. (Poaceae - Andropogoneae - Saccharinae) no Rio Grande do Sul, Brasil). A subtribo Saccharinae pertence à tribo Andropogoneae e inclui cerca de 140 espécies distribuídas em 13 gêneros, entre eles Eriochrysis, Imperata e Saccharum. O presente trabalho consiste em um levantamento das espécies desses três gêneros no estado do Rio Grande do Sul. Foram realizadas coletas intensivas em diferentes regiões fisiográficas do estado, bem como revisão de diversos herbários. Foi confirmada a ocorrência de três espécies nativas de cada um dos gêneros, no Rio Grande do Sul, além da “canade- açúcar” (Saccharum officinarum L.), cultivada no estado. A ocorrência dessas espécies no Rio Grande do Sul, embora já citada em trabalhos anteriores, constitui um acréscimo ao Catálogo de Plantas e Fungos do Brasil, recentemente publicado. A partir de materiais adicionais analisados, Eriochrysis villosa Swallen está sendo citada pela primeira vez para o estado do Paraná. São fornecidas chaves de identificação para as espécies confirmadas, descrições, informações sobre a sua distribuição geográfica, habitat e períodos de florescimento e frutificação, além de ilustrações de caracteres de importância taxonômica. / (The genera Eriochrysis P. Beauv., Imperata Cirillo and Saccharum L. (Poaceae - Andropogoneae - Saccharinae) in Rio Grande do Sul State, Brazil). The subtribe Saccharinae belongs to the tribe Andropogoneae and includes about 140 species, in 13 genera, including Eriochrysis, Imperata and Saccharum. This work consists of a survey of the species of these three genera in Rio Grande do Sul. Intensive field collections were made in different physiographic regions of the State, as well as revision of several herbaria. The occurrence of three native species of each genus was confirmed, in addition to the "sugar cane" (Saccharum officinarum L.), that is cultivated in the area. The occurrence of these species in Rio Grande do Sul, although mentioned in previous studies, should be added to the recently published Catalogue of Plants and Fungi of Brazil. Based on additional material examined, Eriochrysis villosa Swallen is being reported for the first time to Paraná State, Brazil. Analytical keys for the identification of the confirmed species, descriptions, data on their geographical distribution, habitat and periods of flowering and fruiting, as well as illustrations of important taxonomic characters, are provided.