Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Biopolymers and bio-degradable polymers are of utmost importance to ensure a sustainable economy. Industry depends on raw material, which in many cases are derived from fossil fuels, but in light of looming energy crises and green revolutions attention is being directed at cellulose and starch biopolymers. This study was therefore set forth to investigate novel genetic key elements of cell wall metabolism in Arabidopsis thaliana and starch synthesis in an under-utilized southern African crop plant, Tylosema esculentum. In the first section of the study a cDNA library of good quality was constructed from regenerating A. thaliana protoplasts as it was expected to be enriching for genes involved in cell wall biosynthesis. Small scale EST sequencing of the library confirmed that a few sequences were similar to genes identified to be highly expressed during protoplast regeneration. The library was to be screened by expression in a microalgae as it is anticipated that cell wall metabolising genes would change the wall structure and visibly alter the colony morphology. An attempt was made at establishing a high-throughput transformation system in the unicellular algae Chlorella protothecoides in which the library was proposed to be screened. Conventional microalgal transformation techniques do not appear to be effective in this strain as the study produced no transgenic algae. Alternative studies into a screening system within another species could still lead to the identification of cell wall biosynthetic genes, which was the first objective in the study. The second objective in the study was to investigate the potential of the orphan crop T. esculentum as starch-producing cash-crop in developing southern African countries. In this section of the study a cDNA library of good quality was produced form the tuber of T. esculentum. The library was transferred to an expression vector and screened functionally in E. coli for the presence of sequences with starch synthase activity. No sequences have been identified yet and screening procedures are still on-going. The starch content in the tuber has also been determined for the first time. The relatively high starch content in combination with low agricultural inputs indicate the potential of the plant as an industrial starch source. Further investigations into the nature of the starch are proposed to identify prospective buyers within the industry. / AFRIKAANSE OPSOMMING: Biopolimere en bio-afbreekbare polimere is van kardinale belang om ‘n volhoubare ekonomie te ontwikkel. Industriële toepassings maak op die oomblik hoofsaaklik staat op fossielbrandstof verwante bronne, maar met die oog op ‘n groen revolusie en energie krissise wat dreig word meer belangstelling getoon in sellulose en stysel biopolimere. Hierdie studie is daarom onderneem om genetiese elemente te identifiseer wat betrokke is by die sintese van die selwand in Arabidopsis thaliana en stysel sintese in die suider Afrikaanse gewas Tylosema esculentum wat grotendeels onderbenut is. In die eerste deel van die studie is ‘n cDNA biblioteek, van goeie kwaliteit, geskep vanuit A. thaliana protoplaste wat besig was om hulle selwande te herbou. Dit word verwag dat die protoplaste gedurende die tydperk aktief besig sal wees om gene uit te druk wat betrokke is by selwandsintese. DNA volgordebepaling het bevestig dat ‘n klein aantal volgordes ooreengestem het met gene wat voorheen gevind was om in ‘n oormaat uitgedruk te word tydens die herbou van protoplas-selwande. Daar was beoog om die biblioteek in ‘n mikroalge uit te druk en sodoende die morfologie op kolonievlak waar te neem vir verandering wat in die selwand meegebring is. Om hierdie rede was die doel om ‘n hoë opbrengs transformasie sisteem te ontwikkel in die mikroalge Chlorella protothecoides. Algemene mikroalge transformasie tegnieke blyk om nie effektief in die spesie te wees nie aangesien geen transgeniese alge waargeneem is nie. Die ontwikkeling van ‘n soortgelyke proses in ‘n ander spesie kan steeds lei na die ontdekking van gene betrokke by selwandsintese in A. thaliana wat die eerste uitkoms van die projek as geheel was. Die tweede uitkoms van die projek was om te ondersoek wat die waarskynlikheid was om T. esculentum te kommersialiseer as ‘n stysel gewas en sodoende ‘n inkomste te skep vir arm boere in ontwikkelende lande in suider Afrika. In hierdie gedeelte van die projek was daar ‘n goeie cDNA biblioteek geskep uit die knol van T. esculentum. Die biblioteek is oorgedra na ‘n plasmied waarop dit aktief uitgedruk kon word in Escherischia coli G6MD2 en daar is gesoek na volgordes wat lei na die sintese van stysel in hierdie bakterieë. Tot op hede is geen sulke volgordes gevind nie, maar die ondersoek gaan steeds voort. Die styselinhoud van die knol is ook vir die eerste keer bepaal in hierdie ondersoek. ‘n Styselinhoud wat relatief hoog is en die lae moeite wat geverg word om die gewas te verbou toon dat die plant potensieel het as ‘n kommersiële bron van stysel. Verdere ondersoeke in die aard van die stysel word ook voorgestel om toekomstige industriële kopers te identifiseer.

Thesis (MSc (Genetics. Institute for Plant Biotechnology)--Stellenbosch University, 2008. / Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts of sugars and organic acids present, as well as soluble solid content. This study had three components: 1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars and organic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increased pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis of transgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol or apoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries. Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairly similar with respect to sugar and organic acid content. Twelve cytosolic enzymes were investigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP, pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPase activity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively, due to the purification method employed. These analyses established methodology for the analysis of transgenic berries. Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or to express Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertase construct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed and the presence of each transgene confirmed by PCR. Untransformed Selekta was used as control in both transgenic studies. Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripe fruit. Sugar and organic acid content of ripe berries with high PFP activity was determined. Although berries displayed marked changes in sugar composition, the total sugar content was similar to controls, in all except one line. Organic acid content was decreased, leading to a clear reduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberry fruit. Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibited total invertase activity similar to controls and were not analysed further. Berries with apoplasttargeted SCI displayed three-fold increases in invertase activity compared to controls. Total sugar content was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effect of increased invertase activity on metabolites, maximum catalytic activity of enzymes involved in cytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selected in these studies were subsequently vegetatively replicated and future work will include immature fruit.

Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.

Thesis (MSc (Genetics. Plant Biotechnology)) --Stellenbosch University, 2008. / The main aim of the work presented in this thesis was to further our understanding of the role of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, by specifically investigating its potential contribution to phloem metabolism. PFP activity in sugarcane internodal tissue is inversely correlated to sucrose content across varieties that differ in their sucrose accumulation abilities. This apparent correlation is in contrast to previous studies that suggest PFP plays an insignificant role in metabolism. In the first part of this study an immunological characterisation of the two subunits of sugarcane PFP was conducted to establish whether it differ significantly from other plant species in terms of size and distribution. Both the alpha and beta subunit appears to be approximately sixty kilo Daltons in size and uniform in their relative distribution to each other in the various plant organs of sugarcane. Although the observed alpha subunit size is less than that predicted this could be explained at the hand of post translational modification, in essence the sugarcane PFP subunits appear similar than that described for other plants especially that of tobacco which was employed as a model system later on in this study. The only direct way to investigate PFP’s contribution to phloem metabolism is to alter its activity by recombinant DNA technologies. Therefore, in the second part of the study transformation systems were designed for both the constitutive and phloem specific downand up-regulation of PFP activity. For the down-regulation of activity a post transcriptional gene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencing system, was employed. A partial sequence of the PFP-beta subunit was isolated and used in vector construction. For the over-expression the Giardia lamblia PFP gene was used. The model plant tobacco was employed to investigate PFP’s effect on phloem metabolism and transport of assimilate. Transgene insertion was accomplished by means of Agobacterium mediated transformation and tissue specific manipulation of PFP activity was confirmed by in situ activity staining.

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A two-component transposable element system consisting of a stabilized Activator (Acst) and a chimeric Dissociation (Ds) element has been introduced into the genome of Brassica napus. This Acst/ Ds system incorporates the use of several highly effective screenable and selectable markers. One of these markers is the maize Lc gene, a transcriptional regulator of flavonoid biosynthetic genes. This substrate-independent screenable marker was tested for the first time in B. napus and I show that when overexpressed, there is augmented trichome production and a light-dependent, enhanced accumulation of anthocyanins in B. napus plants. The phenotypes are expressed under a wide range of conditions, are visually distinct, and are observed throughout plant development. When used as a visual marker for the Acst element, Lc B. napus plants were rapidly identified among F2 segregating populations. As part of my goal to develop a very efficient Acst/Ds system for use in B. napus, a conditional negative selectable marker, the E. coli codA gene, was also tested for the first time in B. napus. This was done because use of a substrate-dependent negative selectable marker can facilitate the rapid and reliable identification of stable Ds transposition events when used as a marker for the Acst T-DNA. The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non-toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil. In codA transformed B. napus seedlings, expression of cytosine deaminase results in a severe suppression of growth and this phenotype is dependent on the presence of the 5-FC substrate. Wild-type seedlings, however, lack endogenous cytosine deaminase activity and appear unaffected by the presence of 5-FC in the growth media. These results indicate that codA has the potential to be used effectively in B. napus as a substrate-dependent negative selectable marker for the Acst T-DNA. To determine if Ac transposase cou

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur. / The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%). / The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass. / The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

The post-genomics era has put at the disposal of modern plant breeders an endless list of genetic building blocks for the design of new biotechnological crops. After a first wave of single-gene transgenic with controversial public acceptance, genomic information and technology is paving the way for increasingly complex designs based in multiple gene engineering. Those designs aiming at the production of inexpensive health-promoting compounds are most likely to be welcomed by consumers. In this project we plan to develop new multigene assembling tools. During this PhD, a standardized collection of interchangeable genetic parts (including promoters, CDS, P-DNAs, etc) and vectors will be developed. The collection, inspired in Synthetic Biology standards, will be made easy-to-assemble in an interchangeable, semi-idempotent and seamless fashion by the addition of flanking recognition sites of type IIS Restriction endonucleases. The construction of the collection will facilitate multigene engineering and will constitute a first step towards enabling Synthetic Biology in plants. / Sarrión Perdigones, MA. (2014). Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35399 / TESIS

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