The relative distribution of dry matter over shoot and tuberous roots is an important factor in determining the yield of cassava. Under sub-tropical conditions the dry matter distribution pattern is not always efficient. During the summer the vegetative growth is generally excessive due to long days and high temperatures. Furthermore, it was found that a reduction of tuberous root mass occurs shortly after the winter period when the new canopy is formed. The role of endogenous hormones, in particular abscisic acid and cytokinins, in dry matter distribution and tuberization was studied. Furthermore, experiments were conducted on the effect of exogenous applied plant growth regulators on the growth of cassava. Inhibitor activity present in plant extracts, was tentatively identified as cis-abscisic acid. A high level of inhibitor activity, co-eluting with abscisic acid, was found throughout the tuberous roots. The highest level of inhibitor activity was detected under conditions which caused a high rate of tuberous root growth, ego with a low level of nitrogen fertilization. Gibberellic acid application to the leaves promoted shoot growth and led to a decrease in inhibitor activity of the tuberous roots. Shoot removal, however, stopped tuberous root growth without leading to changes in inhibitor activity. No clear correlation was found between inhibitor activity of tuberous roots and the rate of tuberous root growth of plants harvested from the field tentatively identified as the major cytokinins in tuberous root extracts. Cytokinin activity was concentrated in the meristematic region of the xylem. The level of cytokinin activity in the roots was much higher than in primary roots of the same plants. Gibberellic acid application to the leaves and shoot removal resulted in a reduction of the cytokinin level of tuberous roots. The nitrogen application to the plants had no clear effect on the cytokinin levels. In field-grown plants the highest level of cytokinin activity was found shortly after tuber initiation. Applications of Alar caused satisfactory reduction of shoot growth of young cassava plants grown in the greenhouse. The internodes were shortened, the leaf area generally reduced, while a relatively larger part of the dry matter was allocated to storage roots. The effect of Alar was further studied in field experiments. A growth analysis showed that a reduction of tuberous root mass occured shortly after the winter period, in September. Later in the second growing season, shoot and tuberous root mass increased at a relatively constant rate. Alar application (up to 4,5 grammes per Iitre) early in the second season failed to bring about major changes in dry matter distribution. Shoot removal at the end of the winter period was followed by excessive vegetative growth. Alar application reduced the internode length but the reduction of shoot growth was insufficient to cause a significant increase in yield. A nitrogen topdressing at the start of the second season increased the vegetative growth. However, Alar application, later in the season, did not inhibit shoot growth at any of the nitrogen levels applied. RSW 0411 which caused good reduction of shoot growth in the greenhouse, was not effective in the field. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Cassava flour is recommended for substitution with wheat flour in composite flour for baking. The potential use of cassava flour in the food and pharmaceutical industries is attributed to its gluten-free nature and excellent functional properties. However, optimum packaging solution and storage conditions for cassava flour is critical in maintaining the quality attributes and shelf-life stability during storage. Therefore, this study focused on investigating the effects of package types (plastic buckets, low density polyethylene (LDPE) bags and brown paper bags) and storage conditions (cool condition (15 °C, 90% RH); ambient condition (23 °C, 60% RH); and higher condition (38 °C, 60% RH)) on the functional properties, quality attributes and shelf-life stability of cassava flour (cvs. ‘TME 419’ and ‘UMUCASS 36’) developed at the National Root Crops Research Institute, Umudike, Nigeria. Proximate composition, physicochemical attributes, functional properties, and microbial safety of flour were analysed every 4 weeks for 12 weeks storage duration. Flour stored under cool condition with paper bags became moist and sticky with appearance of mould growth before 4 weeks of storage. However, at the end of 12 weeks storage, a decline in moisture content of 11.00 ± 0.02 and 7.05 ± 0.01% flour of ‘TME 419’ was observed at ambient and higher conditions, respectively. Rate of moisture decline was similar in flour of ‘UMUCASS 36’. A slight decrease in protein content of flour was observed during the 12 weeks storage from 1.9 ± 0.07 to 1.30 ± 0.001% for cv. ‘TME 419’ and 3.0 ± 0.05 to 2.27 ± 0.001% for cv. ‘UMUCASS 36’; however, no significant difference was observed under ambient and higher conditions. Cassava flour packed in paper bags and stored under higher condition (38 °C, 60%) had the highest loss (50%) of carotenoid content from 1.84 ± 0.10% to 0.91 ± 0.08%, while a minimal loss (24%) of carotenoid was observed in flour packed in plastic buckets under ambient condition. The concentration of hydrogen cyanide (HCN) decreased across all treatments and was below the safe cyanide level of 50 µg/ mL for food products. After the 12 weeks of storage, flour packed in plastic buckets had the highest aerobic mesophilic bacterial counts (3.43 ± 0.04 log cfu/ g) followed by flour in LDPE bags (3.37 ± 0.03 log cfu/ g) and paper bags (3.35 ± 0.01 log cfu/ g). No significant difference was observed in the package types; however the counts observed were within the acceptable microbial limit Swelling power (SP), solubility and peak viscosity were used to characterise the changes in functional and pasting properties of cassava flour relevant in food industries. Flour packed in plastic buckets under ambient condition had the lowest swelling power (8.48 ± 0.55%) and peak viscosity (260 ± 0.51 RVU) compared to flour packed in LDPE and paper bags with (9.10 ± 0.13 and 9.32 ± 0.41%) SP and (263.67 ± 4.04 RVU and 302 ± 9.52 RVU) peak viscosity, respectively. The essential minerals (sodium, potassium, copper, and iron) were significantly higher in flour of ‘TME 419’ compared to ‘UMUCASS 36’. In summary, for the production of high grade foods such as bread where higher swelling power and viscosities are required, flour from ‘TME 419’ packed with paper bags under higher condition could be desirable. In addition, for infant formulation, flour from ‘UMUCASS 36’ packed in plastic buckets and stored under ambient condition which best maintained nutritional contents (protein and fat) and had the lowest peak viscosity would be more suitable. Flour from both cassava cultivars could be stored up to 12 weeks duration under ambient and hot tropical conditions using all package types evaluated. However, storage with paper bag under higher condition offers the chances of better shelf -life stability of cassava flour. / AFRIKAANSE OPSOMMING: Daar word aanbeveel dat kassavameel in plaas van koringmeel in saamgestelde meel by gebak gebruik word. Die potensiële gebruik van kassavameel in die kos- en farmaseutiese industrieë word toegeskryf aan die glutenvrye aard en funksionele kenmerke daarvan. Optimale verpakking en stoortoestande is egter belangrik vir die instandhouding van die gehalte kenmerke en raklewe stabiliteit tydens stoor. Daarom is die fokus van hierdie studie op die effek van verskillende tipes verpakking (plastiekemmers, lae densiteits politelien (LDPE) sakke en bruin papiersakke) en stoortoestande (koel toestande (15 °C, 90% RH); omringende temperature (23 °C, 60% RH); en hoër temperature (38 °C, 60% RH) op die funksionele kenmerke, gehalte kenmerke en raklewe stabiliteit van kassavameel (kultivare. ‘TME 419’ en ‘UMUCASS 36’) wat by die Nasionale Wortelgewasse Navorsingsinstituut, Umudike, Nigerië ontwikkel is. Die komposisie, fisiochemiese kenmerke, funksionele kenmerke en mikrobiale veiligheid van meel is elke vier weke tydens die 12-weke stoortydperk ontleed. Meel wat onder koeltoestande in papiersakke gestoor word, word klam en taai en swamme maak by vier weke van stoor ’n verskyning. Teen die einde van 12 weke stoortydperk is daar ’n afname in klammigheid van 11.00 ± 0.02 en 7.05 ± 0.01% in ‘TME 419’ meel by onderskeidelik omgewings- en hoër temperature. Die afname in klammigheid is soortgelyk by ‘UMUCASS 36’ meel. ’n Effense afname in die proteïen inhoud van die meel is tydens die 12- weke stoortydperk vanaf 1.9 ± 0.07 tot 1.30 ± 0.001% by die kultivaar . ‘TME 419’ en 3.0 ± 0.05 tot 2.27 ± 0.001% vir kultivaar ‘UMUCASS 36’ opgemerk. Geen noemenswaardige verskil is egter onder omgewings- en hoër temperature opgemerk nie. Kassavameel wat in papiersakke en onder hoër temperature (38 °C, 60%) gestoor is het die hoogste verlies (50%) aan karotien inhoud vanaf 1.84 ± 0.10% tot 0.91 ± 0.08% getoon , terwyl ’n minimale verlies (24%) by meel wat in plastiekemmers onder omgewingstemperature verpak is, opgemerk is. Die konsentrasie van waterstof hidrosianied (HCN) het tydens alle behandelinge afgeneem en was onder die veilige vlak van 50 µg/ mL vir kosprodukte. Na ’n 12-weke stoortydperk het die meel wat in plastiekemmers verpak is, die hoogste mesofiliese bakterië telling getoon \ (3.43 ± 0.04 log cfu/ g) gevolg deur die meel in die LDPE sakke (3.37 ± 0.03 log cfu/ g) en papiersakke (3.35 ± 0.01 log cfu/ g). Daar was geen merkbare verskil ten opsigte van verpakkingstipes nie; die tellings wat geneem is, was almal binne die aanvaarbare mikrobiale perk. Swelkrag (SP), oplosbaarheid en piek viskositeit is gebruik om die veranderinge in funksionele kenmerke van kassavameel wat betrekking het op die kosindustrie, te ondersoek. Meel wat onder omgewingstemperature in plastiekemmers verpak is, het die laagste swelkrag (8.48 ± 0.55%) en piekviskositeit getoon (260 ± 0.51 RVU) getoon vergeleke met meel wat in LDPE- en papiersakke (9.10 ± 0.13 en 9.32 ± 0.41%) swelkrag en (263.67 ± 4.04 RVU en 302 ± 9.52 RVU) piekviskositeit, onderskeidelik toon. Die belangrike minerale (natrium, kalium, koper en yster) was noemenswaardig hoër in die ‘TME 419’ meel vergeleke met ‘UMUCASS 36’. Ten slotte, vir die produksie van hoëgraad kossoorte soos brood waar hoë swelkrag en viskositeit belangrik is, is In ‘TME 419’ meel onder hoër toestande verpak in papiersakke, die beste keuse. In die geval egter van babakosse is ‘UMUCASS 36’meel wat in plastiekemmers verpak en onder omgewingstemperature gestoor is, en wat dus koswaardes (proteïen en vette) behou en wat die laagste piek viskositeit het, meer geskik. Meel van albei kultivaars kan vir tot twaalf weke onder omgewings- en hoë, tropiese temperature in al die verpakkingstipes wat evalueer is, gestoor word. Stoor in papiersakke onder hoër temperature verbeter egter die kanse op beter raklewe stabiliteit.

Made available in DSpace on 2014-06-11T19:24:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-06Bitstream added on 2014-06-13T20:12:53Z : No. of bitstreams: 1 pinto_phm_me_botfca.pdf: 1393017 bytes, checksum: ba5d604cb5f1d74e2ea4e7db1accf8da (MD5) / Manipueira de extração de fécula de mandioca, em separado da água de lavagem das raízes, através de biodigestores anaeróbios de fluxo ascendente, com separação das fases, sem controle de temperatura ou adição de produtos químicos e, avaliar sua adequabilidade, através das suas características físicas e químicas para lançamento em corpo receptor, sistema público coletor de esgotos ou, aplicação em processo de fertirrigação. Depois dos reatores estabilizados, foram realizados ensaios variando a vazão de alimentação com 8,0; 12,0 e 16,0 Ld-1, correspondentes a um tempo de retenção hidráulica de 8,17; 5,44 e 4,08 dias respectivamente. Os melhores resultados para redução da carga orgânica foram obtidos com os tempos de retenção hidráulica (TRH) de 8,17 e 5,44 dias com eficiências médias de 89,8 e 80,9% respectivamente. As características físicas e químicas dos efluentes tratados foram comparadas com os valores estabelecidos na legislação estadual, federal e, com os parâmetros utilizados pelo órgão ambiental fiscalizador. Os resultados obtidos mostraram que o efluente tratado atende parcialmente aos requisitos legais para o lançamento em corpos receptores, devido ao teor elevado de nitrogênio amoniacal. Por outro lado, foram atendidos integralmente os requisitos legais para o lançamento na rede pública coletora de esgotos. O efluente tratado não atendeu às recomendações requeridas pelo órgão ambiental fiscalizador para a sua disposição através de processo de fertirrigação devido aos teores elevados de ferro (Fe++) e de fluoretos (F-). Considerando os resultados obtidos, concluímos que, devido à simplicidade do sistema utilizado, com a implantação de melhorias como um pós tratamento, poderiam ser atingidos os parâmetros que atenderiam integralmente a legislação. / The aim of this paper was to evaluate the efficiency of the treatment of cassava wastewater, separately from the root washing water, by means of ascending flux anaerobic biodigesters, with separation of the phases, without temperature control or addition of chemical products and to evaluate its suitability by means of its physical and chemical characteristics for throwing in receiving body, public sewage system or application in fertilization and irrigation. After reactors had been stabilized, essays were conducted varying feeding flow with 8.0, 12.0 and 16.0 Ld-1, corresponding to a hydraulic retention time of 8.17, 5.44 and 4.08 days, respectively. The best reduction for organic load reduction were obtained with hydraulic retention times (HRT) of 8.17 and 5.44 days with mean efficiencies of 89.8 and 90.9%, respectively. Physical and chemical characteristics of treated effluents were compared with the values established in the current state and federal legislation and with the parameters used by the environmental supervising organ. The results we obtained showed that the treated effluent partially meets the legal requirements for throwing in receiving bodies owing to high contents of ammonia nitrogen. On the other hand, legal requirements were fully met to throw effluents into the public sewage system. The treated effluent did not fulfill the recommendations required by the environmental supervising organ for its disposal by means of fertilization and irrigation due to high concentrations of iron (Fe++) and fluorides (F-). Considering the results obtained, we conclude that, due to the simplicity of the system used, as improvements such as after-treatment are made, parameters fully obeying the legislation could be met.

A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016 / Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome. / LG2017

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Cassava mosaic disease (CMD) is caused by whitefly-transmitted geminiviruses and is a major constraint to cassava production in Africa. Field surveys were conducted in three (Bushbuckridge, Mariti and Tonga) cassava growing areas of Limpopo and Mpumalanga provinces in South Africa during two seasons (2004/2005 and 2005/2006). Results showed that a higher percentage (27.1%) of CMD infection was due to the use of infected planting materials compared to whitefly borne-infections (10.4%). Disease symptoms were generally mild. There was no change in disease incidence over the survey period. Molecular characterization of cassava mosaic geminiviruses (CMG’s), using differential primer PCR, restriction fragment length polymorphisms (RFLP’s), phylogenetic and recombination analysis and screening for satellite DNA’s. Differential primer PCR and RFLP’s showed that African cassava mosaic virus (ACMV) was the most prevalent virus in South Africa and that mixed infections were a common occurrence. Phylogenetic analysis and RFLP’s showed the presence of a ‘new’ strain of ACMV in South Africa. EACMV isolates from this study showed more frequent recombination compared to ACMV isolates. None of the samples tested positive for satellite DNA’s. Phylogenetic analysis of Bemisia tabaci using the mitochondrial cytochrome oxidase gene sequences revealed a ‘new’ sister clade of B. tabaci that is closely related to the previously identified southern African clade and the presence of the Q biotype that groups with Q biotypes of North African/Mediterranean origin. Good cultural practices, introduction of resistant cultivars and continuous monitoring are required to reduce the impact of CMD in South Africa.

This is an interdisciplinary research project on cassava (Manihot esculenta Crantz, Euphorbiaceae) ultimately working towards producing cassava roots which are long-lasting, free of post-harvest physiological deterioration (PPD). It aims to contribute to ensuring food security. In cassava, scopoletin and its -glycoside scopolin are considered phytoanticipants, not phytoalexins, due to their increasing accumulation during the PPD process compared to their barely detectable levels in fresh roots. Starting with a focussed literature review on the potential of cassava, contrasted with its limitations on harvesting due to PPD, and biosynthesis along the phenylpropanoid pathway of key hydroxycoumarins, e.g. scopoletin and esculetin, the associated gaps in our current knowledge have been set out. Whether scopoletin is biosynthesized de novo from L-phenylalanine in response to stress, or whether stress prompts its release from the corresponding glycoside is unknown. Therefore, assessing hydroxycoumarin biosynthesis and quantifying their accumulation patterns have been undertaken in wild-type and transgenic plants in order to elucidate the divergence in scopoletin biosynthetic pathways. The identification of key genes on each pathway leading to scopoletin in cassava, and then exploring their functional identities using the model plant A. thaliana and genetically engineered E. coli, where the genes were isolated, cloned, and expressed, were also undertaken. Transgenic A. thaliana lines with no activity of the key enzymes on the proposed pathway, namely F6ʹH1, CCoAOMT, and EOMT, were developed. Competition feeding experiments using stable isotopically labelled potential biosynthetic intermediates showed the incorporation of labelled ferulate into scopoletin in transgenic A.t-F6´H1 and M.e-F6´H. This confirmed the activity of other hydroxylase enzymes rather than F6´H1 in the ortho-hydroxylation steps. The hydroxycoumarins of interest were isolated, characterized, and quantified in the wild type and mutant lines using chromatographic and spectroscopic techniques, mainly NMR, HR-MS, and LC-MS. Taken together, a significant contribution to knowledge about hydroxycoumarin biosynthesis has been made.

Thesis (Ph. D.)--Washington State University, December 2009. / Title from PDF title page (viewed on Dec. 11, 2009). "Department of Plant Pathology." Includes bibliographical references.