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Yip Mei-kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-102). / Abstracts in English and Chinese. / Acknowledgments --- p.i / List of abbreviations --- p.ii / Abstract --- p.iii / Table of Contents --- p.vi / List of tables --- p.x / List of figures --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Plant of interest --- p.1 / Chapter 1.2 --- Literature review --- p.2 / Chapter 1.2.1 --- Anthocyanins-natural pigments in plants --- p.2 / Chapter 1.2.1.1 --- Sources and biosynthesis --- p.2 / Chapter 1.2.1.2 --- Chemical properties --- p.2 / Chapter 1.2.1.3 --- Biological effects --- p.3 / Chapter 1.2.2 --- Characterization of anthocyanins --- p.4 / Chapter 1.2.3 --- Plant tissue and cell cultures --- p.6 / Chapter 1.2.4 --- Induction of anthocyanins in plant tissue culture --- p.7 / Chapter 1.2.5 --- Factors affecting anthocyanin production --- p.8 / Chapter 1.2.5.1 --- Plant hormones --- p.8 / Chapter 1.2.5.2 --- Nutrients --- p.9 / Chapter 1.2.5.2.1 --- Phosphate --- p.9 / Chapter 1.2.5.2.2 --- Nitrogen --- p.9 / Chapter 1.2.5.3 --- Osmoticums --- p.10 / Chapter 1.2.5.3.1 --- Sucrose --- p.10 / Chapter 1.2.5.3.2 --- Other factors --- p.10 / Chapter 1.3 --- Research objectives --- p.12 / Chapter 2. --- Materials and methods --- p.16 / Chapter 2.1 --- Plant materials --- p.16 / Chapter 2.2 --- Study of pigment formation at different developmental stages --- p.16 / Chapter 2.2.1 --- Cultivation of Vigna sinensis --- p.16 / Chapter 2.2.2 --- Sample collection --- p.16 / Chapter 2.2.3 --- HPLC analysis of pigmented vegetative tissues --- p.16 / Chapter 2.2.4 --- HPLC analysis of seed coats at different developmental stages --- p.17 / Chapter 2.3 --- Characterization of seed coat pigments --- p.17 / Chapter 2.3.1 --- Extraction of seed coats pigments --- p.17 / Chapter 2.3.2 --- Acid hydrolysis of anthocyanins --- p.17 / Chapter 2.3.3 --- High performance liquid chromatography --- p.18 / Chapter 2.3.3.1 --- HPLC system --- p.18 / Chapter 2.3.3.2 --- Analytical conditions --- p.18 / Chapter 2.4 --- Establishment of tissue culture system --- p.19 / Chapter 2.4.1 --- Aseptic plant stocks --- p.19 / Chapter 2.4.2 --- Shoot-tip cultures --- p.19 / Chapter 2.4.3 --- Callus initiation --- p.19 / Chapter 2.4.3.1 --- From seed coats --- p.20 / Chapter 2.4.3.2 --- From vegetative tissues --- p.20 / Chapter 2.4.3.3 --- Light and dark --- p.20 / Chapter 2.4.4 --- Optimization of callus growth --- p.21 / Chapter 2.4.4.1 --- Basal medium --- p.21 / Chapter 2.4.4.2 --- Combination of various plant hormones --- p.21 / Chapter 2.4.4.3 --- Basal salt --- p.21 / Chapter 2.5 --- Studies of anthocyanin production in hypocotyl callus cultures --- p.22 / Chapter 2.5.1 --- Effects of nutrients --- p.22 / Chapter 2.5.1.1 --- Nitrogen --- p.22 / Chapter 2.5.1.2 --- Phosphate --- p.22 / Chapter 2.5.2 --- Osmotic stress --- p.22 / Chapter 2.5.2.1 --- Sucrose --- p.22 / Chapter 2.5.2.2 --- Mannitol --- p.23 / Chapter 2.5.2.3 --- Sodium chloride --- p.23 / Chapter 2.5.2.4 --- Polyethylene glycol --- p.23 / Chapter 2.6 --- Studies of anthocyanin production in cell suspension cultures --- p.23 / Chapter 2.6.1 --- Effects of nutrients --- p.24 / Chapter 2.6.1.1 --- Nitrogen --- p.24 / Chapter 2.6.1.2 --- Phosphate --- p.24 / Chapter 2.6.2 --- Osmotic stress --- p.25 / Chapter 2.6.2.1 --- Sucrose --- p.25 / Chapter 2.6.2.2 --- Polyethylene glycol --- p.25 / Chapter 2.6.3 --- Effects of other factors --- p.25 / Chapter 2.6.3.1 --- Riboflavin --- p.25 / Chapter 2.6.3.2 --- pH --- p.26 / Chapter 2.7 --- Measurement of cell growth --- p.26 / Chapter 2.8 --- Estimation of anthocyanins --- p.26 / Chapter 2.9 --- Statistical analysis --- p.27 / Chapter 3. --- Results --- p.30 / Chapter 3.1 --- Study of pigment formation at different developmental stages --- p.30 / Chapter 3.1.1 --- General description --- p.30 / Chapter 3.1.2 --- HPLC analysis of developing seed coats and other vegetative tissues --- p.30 / Chapter 3.1.3 --- The relationship between pigment formation and seed development --- p.30 / Chapter 3.2 --- Characterization of seed coat pigments --- p.31 / Chapter 3.3 --- Establishment of tissue culture system --- p.43 / Chapter 3.3.1 --- Callus initiations from seed coats --- p.43 / Chapter 3.3.2 --- Callus initiations from vegetative tissues --- p.43 / Chapter 3.3.3 --- Optimization of callus growth --- p.43 / Chapter 3.3.3.1 --- Effects of NAA and BA --- p.43 / Chapter 3.3.3.2 --- Effects of basal medium and combinations of plant hormones --- p.44 / Chapter 3.3.3.3 --- Effects of basal salt --- p.44 / Chapter 3.4 --- Studies of anthocyanin production in hypocotyl callus culture --- p.54 / Chapter 3.4.1 --- Effects of nutrients --- p.54 / Chapter 3.4.1.1 --- Effects of total nitrogen --- p.54 / Chapter 3.4.1.2 --- Effects of phosphate --- p.54 / Chapter 3.4.2 --- Effects of plant hormones --- p.55 / Chapter 3.4.3 --- Osmotic stress --- p.55 / Chapter 3.5 --- Establishment of suspension culture system --- p.64 / Chapter 3.6 --- Studies of anthocyanin production in seed coat suspension cultures --- p.64 / Chapter 3.6.1 --- Nutrient effects on suspension cultures --- p.64 / Chapter 3.6.2 --- Osmotic stress on suspension cultures --- p.65 / Chapter 3.6.3 --- Effects of phosphate with high nitrogen --- p.65 / Chapter 3.6.4 --- Effects of riboflavin with high nitrogen --- p.66 / Chapter 3.6.5 --- Influence of pH with high nitrogen --- p.66 / Chapter 4. --- Discussion --- p.79 / Chapter 4.1 --- Anthocyanin in vegetative tissues and seed coats of Vigna sinensis --- p.79 / Chapter 4.2 --- Factors affecting callus initiation in Vigna sinensis --- p.81 / Chapter 4.2.1 --- Explant types --- p.81 / Chapter 4.2.2 --- Plant hormones --- p.82 / Chapter 4.2.3 --- Basal medium --- p.82 / Chapter 4.3 --- Factors affecting anthocyanin production in callus cultures derived from hypocotyls --- p.83 / Chapter 4.3.1 --- Nutrients --- p.83 / Chapter 4.3.2 --- Osmotic stress --- p.85 / Chapter 4.4 --- Factors affecting anthocyanin production in suspension culture derived from seed coats --- p.86 / Chapter 4.4.1 --- Nutrients --- p.86 / Chapter 4.4.2 --- Osmotic stress --- p.87 / Chapter 4.5 --- Comparison of anthocyanin production from natural source and plant tissue cultures of V.sinensis --- p.89 / Chapter 4.6 --- Further studies --- p.89 / Chapter 5. --- Conclusions --- p.91 / References --- p.93

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Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2010 / A 29 kDa lectin that shows specificity for galactose was isolated from Lonchocarpus capassa seeds by a combination of ammonium sulphate precipitation and affinity chromatography on a galactose-sepharose column. The 29 kDa lectin subunit co-purified with a 45 kDa subunit. The N-terminal sequence of the 29 kDa subunit showed homology to other legume lectins while that of 45 kDa subunit was capped. A 360 bp fragment was amplified using degenerate primers designed from internal protein sequences of the 29 kDa subunit and a 5´ RACE system primer. The cDNA fragment was cloned into pTz57R/Tvector and transformed into E. coli. The partial amino acid sequence of the lectin subunit was deduced from the nucleotide sequence of the clone. The 360 bp fragment consisted of 342 bp sequence coding for the start codon, leader sequence, N-Terminal sequence and sequences of the 79 amino acids from N-terminus. Comparison of the deduced amino acid sequence with other legume lectins showed regions of sequence homology with precursor sequences of Robinia pseudoacacia Bark lectin, a non seed lectin from Pisum sativum (pea), and the galactose specific peanut agglutinin (PNA) from Arachis hypogaea. Alignment of these sequences showed conserved regions including the metal binding sites found in all legume lectins. The 5´ end DNA sequence was used to design locus-specific primers which were used with genome walking cassette primers in an attempt to amplify the full L. capassa lectin gene. The cassette primers were designed from restriction enzyme sites on the cassette. Of all the restriction enzymes on the cassette Hind III and the L. capassa gene-specific primers amplified 288 bp of the 342 bp sequence already obtained from sequencing of the cDNA sequence with minor amino acid differences. Although the full lectin sequence was not obtained the study confirmed the presence of a galactose-specific lectin in L. capassa seeds.

In arid end semiarid areas in the Western United States, soluble salts tend to accumulate in the soil in amounts harmful to crop production. A considerable portion of this land cannot be reclaimed due to the poor quality of permeability of the soil o Therefore, the wise selection of crops that will produce satisfactory yields on these soils in necessary.

Phytophthora medicaginis is an important soil-borne oomycete pathogen of lucerne (Medicago sativa) and chickpea (Cicer arietinum) within Australia and overseas. To understand the host/pathogen interaction, a pathosystem was developed using the model legume Medicago truncatula. Using the resources developed for genetics and molecular characterisation in this model plant, the aim of this research was to understand the interaction between M. truncatula and P. medicaginis, with a view to improving resistance to this important pathogen in related legumes. To observe and characterise the interaction between M. truncatula and P. medicaginis, a pathosystem was developed by first screening a germplasm collection of 99 M. truncatula accessions. This revealed a continuous distribution in disease phenotypes with variable extremes in natural resistance to P. medicaginis culture UQ5750, isolated originally from M. sativa. P. medicaginis zoospore inoculation of 1-2 week-old seedlings in glasshouse experiments proved to be a robust and repeatable method to consistently confirm the responses observed for six key M. truncatula accessions; SA8618 and SA8623 exhibit high natural resistance to this pathogen, accession A17 is moderately resistant, A20 is moderately susceptible and accessions Borung and SA30199 are susceptible. To characterise the genetic basis of resistance to P. medicaginis, two reciprocal F2 populations from cross pollinations between A17 and Borung and SA8618 and SA30199 were produced and then phenotyped for disease symptoms. Genetic segregation patterns indicated the involvement of a gene with a major effect in both reciprocal populations. In particular, a 3:1 segregation ratio for resistance in the F2 populations from cross pollinations between A17 and Borung indicated the possibility of a single dominant gene for moderate resistance. Further phenotyping of F3 families is required to verify this. A M. truncatula linkage map was constructed using 50 F2 individuals of the A17 X Borung population and 49 F2 individuals from the Borung X A17 population. The map, covering 519.3 cM, is comprised of 84 SSR markers with an average distance between markers of 8.7 cM. These are evenly spaced over 7 linkage groups, including a super linkage group conferred by a translocation event between LG4 and LG8 of accession A17. Quantitative trait locus (QTL) analysis confirmed there was a QTL with a major effect in the A17/Borung reciprocal populations. A significant QTL was determined by quantifying two symptoms of P. medicaginis infection - proportion of dead/chlorotic leaves and root fresh weight. The trait loci for both symptoms were located on the same linkage group within the same region, supporting the putative position of the QTL and the authenticity of its involvement in resistance to P. medicaginis. This QTL was located on LG6 and accounted for 69.5% of the observed variation in proportion of dead/chlorotic leaves or 38.1% of the variation in root fresh weight within the inoculated populations. The effect of this QTL on resistance to P. medicaginis translated into 27.5% less dead/chlorotic leaves or 0.86 g more root fresh weight. Other QTLs with minor effects that are potentially involved in the interaction are located elsewhere on LG6 and LG2. However, the marker density of the linkage map and the population size need to be increased to verify this. In parallel to this, an F7 recombinant inbred line (RIL) population of chickpea (BG212 X Jimbour), developed by breeders at the New South Wales Department of Primary Industries (NSW DPI), was also assessed for the genetic basis of resistance to P. medicaginis. Variance component analysis of phenotype scores for this intraspecific RIL population indicated that 57.15% of the differences in between-family and withinfamily variance could be attributed to a genetic component. However, gene-based markers developed in M. truncatula and established simple sequence repeat (SSR) markers of chickpea were not sufficiently polymorphic in size to produce a linkage map for further QTL analysis. An interspecific cross between C. arietinum and C. echinospermum (Howzat X ILWC246) was also performed by breeders at the NSW DPI to develop RILs. In the duration of this research these interspecific RILs were bred to generation F3 and phenotyping assessment had not been performed. However, marker screening of the parents revealed 122 size polymorphic chickpea SSR markers. A sufficient linkage map could be produced for QTL analysis once field assessment of this population is performed. Initial screening of the M. truncatula gene-based markers on the parents of this interspecific cross also revealed that 50% show a sequence-identified base pair difference. A chickpea linkage map incorporating these markers could be comparatively mapped with M. truncatula. Molecular investigations of the M. truncatula/P. medicaginis pathosystem were performed to elucidate the possible underlying defence mechanisms involved in the observed resistance. To determine the function of ethylene in the resistant response, the characterisation of defence associated mutants of M. truncatula and Agrobacterium rhizogenes-mediated ‘hairy root’ transformations were employed. Comparison of response to inoculation of an ethylene insensitive mutant of M. truncatula (sickle) with the moderately resistant background genotype A17 showed that sickle was hypersensitive to P. medicaginis. This indicated that ethylene insensitivity was not the source of resistance to this pathogen and importantly that ethylene is a key defence signalling molecule in the moderate resistance of A17 to P. medicaginis. Agrobacterium-mediated ‘hairy root’ transformations of M. truncatula with 4GCC::Luc constructs, revealed that the production of ethylene and consequently ethylene response factors (ERFs) after inoculation by P. medicaginis was a general defence reaction by all accessions. The two susceptible M. truncatula accessions exhibited a much stronger and earlier response to inoculation than the highly resistant and moderately resistant accessions. This indicated that the resistant response may be directed by a transcriptional component governed by the host genotype, downstream of ethylene production. The M. truncatula/P. medicaginis ‘hairy root’ transformation assay has scope to be a powerful functional genomics tool for this pathogen interaction. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) was employed to determine the general patterns of gene expression and function underlying the response to P. medicaginis infection. Relative changes in gene expression of key enzymes in each of the salicylic acid, jasmonic acid, ethylene and isoflavonoid defence pathways and in genes encoding downstream target proteins revealed potential genes involved in the resistance to P. medicaginis. There was a distinct molecular difference in the response between the high and moderately resistant M. truncatula phenotypes to this pathogen. Moderate resistance to P. medicaginis in M. truncatula is possibly mediated by ethylene and involves the considerable induction of pathogenesis related protein 5 (PR5), which was not the same defence response that conferred the high resistance to P. medicaginis. Early and consistent expression of genes encoding key enzymes of the isoflavonoid pathway by the highly resistant accession indicated that phytoalexin response could be associated with the high resistance. Confirmation of the involvement of isoflavonoid phytoalexins in the high resistance response to P. medicaginis merits further investigation.

In Lombok Island (Indonesia), the ratio of land area to population is already low, and is likely to decrease because of the increasing size of the human population. The management of land resources is, therefore, important, to ensure the wise and sustainable use of the available land in meeting population demands, especially for food. Geographic Information Systems (GIS) have been used successfully in resource management, and this area of their application has been a major driver in the development of GIS. Because agrosystems need to be tuned to the specific characteristics of regional environments, regionalisation is one way to improve agricultural production and the management of agrotechnology development. The identification of potential areas for growing soybean, peanut and common bean was conducted based on two tools: GIS analysis and the 30 group regionalisation. Both techniques have advantages and disadvantages. Using GIS, exact mapping of the potential category of each grid cell can be done, but it cannot be used to estimate the total humid periods and suitable planting times in a year. Using a regionalisation, these can be done, but only when purposes is suggested as more widely applicable than using GIS analysis. Both techniques have a role to play. Based on an initial validation of the techniques employed and the results obtained, further work is suggested, either for the optimum application of the results presently obtained or for the improvement of the techniques of analysis and thence the production of results for future use. / Master of Science (Hons)

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>

Thesis (M. Sc.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Nov. 25, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Plant Science, Department of Agricultural, Food and Nutritional Science, University of Alberta." Includes bibliographical references.