published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

De novo shoots, roots, and flower buds were regenerated on thin cell layer explants excised from pedicel tissue of tomato. Direct shoot organogenesis was greatest when media contained 10µM kinetin and 0.001µM indole-3-acetic acid (IAA); however, shoot regeneration was increased in subsequent experiments by substituting 10 µM zeatin or 10 µM benzyladenine for kinetin. Root formation occurred when media contained higher (0.1 and 10µM) auxin concentrations. Flowers were formed on elongated shoots with several leaves when media contained 10µM IAA and 0.1µM kinetin. Competence for de novo shoot morphogenesis was tested on thin cell layers of eleven tomato cultivars. All tomato cultivars formed shoots directly on thin cell layer explants at varying frequencies (29%-63%). The mean number of shoots per explant was greatest for 'Large Red Cherry', 'Ohio 7814' and 'BL 6807', and poorest for 'Campbell 1327' and 'Red Alert'. Active cell divisions were observed in subepidermal cells during the first week of culture, and meristematic centers of dividing cells were evident after 2 weeks. Well developed shoot apices were observed on 50% of the explants 4 weeks after culture initiation. Shoot morphogenesis was compared among tomato plants placed into micropropagation, callus, and thin cell layer tissue culture systems. More shoots were produced on thin cell layer explants than on cotyledon calli, or micropropagated shoot tips. Genetic recombination rates and map distances were compared among hybrid plants grown in the greenhouse and regenerated from the aforementioned tissue culture systems. Increased recombination rates and map distances were detected between the sunny (sy) and baby leaf syndrome (bls) genes on chromosome 3, and between the white virescence (wv) and anthocyanin reduced (are) genes on chromosome 2. The percent change in the former ranged from 4.5%-5.9% for micropropagated shoot tips, 3.7%-8.5% for plants from cotyledon calli and 2.8%-5.9% for plants from thin cell layers. The percent change between the wv and are loci ranged from 4.5%-6.1% for micropropagated shoot tips, and 3.2%-5.0% and 3.9%-5.7% for plants from cotyledon calli and thin cell layers, respectively. Conversely, a decreased map distance was observed between bls and the solanifolia (sf) locus which is more distal to the centromere on the same arm of chromosome 3 as bls. Changes in recombination rates among plants regenerated from tissue culture may result from an influence of the tissue culture process on meiosis of regenerated plants. / Ph. D.

Suen Pui Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-168). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / List of Figures --- p.vii / List of Tables --- p.ix / List of Abbreviations --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Phosphorus and Plants --- p.3 / Chapter 2.1.1 --- Importance of phosphorus --- p.3 / Chapter 2.1.2 --- Phosphorus is a limiting nutrient --- p.3 / Chapter 2.2 --- Responses of Plants to Phosphate Deficiency --- p.4 / Chapter 2.2.1 --- Morphological changes of plants during phosphate deficiency --- p.5 / Chapter 2.2.1.1 --- Modification of the root system --- p.5 / Chapter 2.2.1.2 --- Symbiotic association of roots with mycorrhiza --- p.6 / Chapter 2.2.2 --- Maintenance of phosphate levels in plants during phosphate deficiency --- p.7 / Chapter 2.2.2.1 --- Phosphate homeostasis in plants --- p.7 / Chapter 2.2.2.2 --- "Enhancement of Pi scavenging, recycling and uptake" --- p.9 / Chapter 2.2.2.3 --- Pi-limited metabolism --- p.11 / Chapter 2.2.3 --- Hormones and phosphate starvation responses --- p.12 / Chapter 2.2.4 --- Regulation of gene expression during phosphate starvation --- p.14 / Chapter 2.2.4.1 --- The pho regulon in bacteria and yeast --- p.14 / Chapter 2.2.4.2 --- The coordination of phosphate starvation induced genes in plants --- p.19 / Chapter 2.2.4.3 --- Signaling phosphate starvation --- p.19 / Chapter 2.2.4.4 --- Phosphite and phosphate starvation --- p.21 / Chapter 2.2.4.5 --- Transcriptional regulation during phosphate starvation --- p.22 / Chapter 2.3 --- Acid Phosphatases in Higher Plants --- p.26 / Chapter 2.3.1 --- Enzymatic properties of acid phosphatases --- p.26 / Chapter 2.3.2 --- Localization and function of acid phosphatases --- p.27 / Chapter 2.3.3 --- Expression of acid phosphatases --- p.28 / Chapter 2.4 --- Purple Acid Phosphatases --- p.29 / Chapter 2.4.1 --- Properties of purple acid phosphatases --- p.29 / Chapter 2.4.2 --- Regulation and expression of plant purple acid phosphatase --- p.32 / Chapter 2.5 --- Tomato Purple Acid Phosphatases --- p.33 / Chapter 2.6 --- Promoter Analysis --- p.35 / Chapter 2.6.1 --- Structure of an eukaryotic promoter --- p.35 / Chapter 2.6.2 --- Promoter analysis by deletion mapping --- p.37 / Chapter 2.6.3 --- The computational approaches in promoter analysis --- p.38 / Chapter 2.6.4 --- Transient expression assay and transgenic expression assay --- p.39 / Chapter 2.7 --- Transcriptional Regulation of Tomato Purple Acid Phosphatase Expression --- p.40 / Chapter 2.8 --- Hypothesis --- p.41 / Chapter Chapter 3: --- Materials and Methods --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Materials --- p.44 / Chapter 3.2.1 --- Chemicals --- p.44 / Chapter 3.2.2 --- Plant materials --- p.44 / Chapter 3.2.3 --- Plasmid vectors and bacterial strains --- p.44 / Chapter 3.2.4 --- Primers design --- p.45 / Chapter 3.2.5 --- Confirmation of sequence fidelity --- p.46 / Chapter 3.3 --- Cloning of the TPAP1 Promoter Fragments --- p.46 / Chapter 3.3.1 --- Genomic DNA extraction --- p.46 / Chapter 3.3.1.1 --- Materials --- p.46 / Chapter 3.3.1.2 --- Procedures --- p.47 / Chapter 3.3.2 --- Cloning strategy of TPAP1 promoter --- p.47 / Chapter 3.3.3 --- TPAP1 promoter cloning --- p.48 / Chapter 3.3.3.1 --- Long-distance PCR --- p.48 / Chapter 3.3.4 --- Chimeric gene constructs --- p.48 / Chapter 3.3.4.1 --- Chimeric gene construction for particle bombardment --- p.51 / Chapter 3.3.4.2 --- Chimeric gene construction for tobacco transformation --- p.51 / Chapter 3.4 --- Transient Expression Assay of the TPAP1 Promoter Fragments --- p.54 / Chapter 3.4.1 --- TPAP1 promoter activity assay --- p.54 / Chapter 3.4.2 --- Preparation of MS culture medium --- p.54 / Chapter 3.4.3 --- Growing tomato seedlings in MS liquid medium --- p.56 / Chapter 3.4.4 --- Biolistic bombardment --- p.56 / Chapter 3.4.5 --- GUS histochemcial staining --- p.57 / Chapter 3.4.5.1 --- Materials --- p.57 / Chapter 3.4.5.2 --- Procedures --- p.57 / Chapter 3.5 --- Transgenic Assay of the TPAP1 Promoter Fragments --- p.58 / Chapter 3.5.1 --- Materials for tobacco transformation --- p.58 / Chapter 3.5.2 --- Agrobacterium tumefaciens preparation --- p.58 / Chapter 3.5.3 --- Tobacco transformation and regeneration --- p.59 / Chapter 3.5.4 --- Promoter activity analysis --- p.60 / Chapter 3.5.4.1 --- Materials --- p.60 / Chapter 3.5.4.2 --- Procedures --- p.60 / Chapter 3.5.5 --- Southern blot analysis --- p.61 / Chapter 3.5.6 --- RNA isolation --- p.61 / Chapter 3.5.6.1 --- Materials --- p.61 / Chapter 3.5.6.2 --- Procedures --- p.61 / Chapter 3.5.7 --- Northern blot analysis --- p.62 / Chapter 3.6 --- Biochemical Analysis of Acid Phosphatase Activities --- p.63 / Chapter 3.6.1 --- Excretion of acid phosphatase into the environment --- p.63 / Chapter 3.6.2 --- Growing tomato seedlings in MS medium --- p.63 / Chapter 3.6.3 --- Acid phosphatase activity assay by p-nitrophenyl phosphate --- p.64 / Chapter 3.6.4 --- Activity-gel detection --- p.65 / Chapter 3.6.4.1 --- Materials --- p.65 / Chapter 3.6.4.2 --- Procedures --- p.65 / Chapter 3.7 --- "Sequence Analysis of the TPAP1 gene, cDNA and promoter" --- p.66 / Chapter 3.7.1 --- Isolation of TPAPl cDNA --- p.66 / Chapter 3.7.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.66 / Chapter 3.7.1.2 --- RT-PCR --- p.67 / Chapter 3.7.2 --- Isolation of TPAP1 gene --- p.67 / Chapter 3.7.2.1 --- PCR amplification of the TPAP1 gene --- p.67 / Chapter 3.7.2.2 --- TPAP1 gene sequence determination --- p.68 / Chapter 3.7.3 --- Sequence analysis --- p.69 / Chapter 3.8 --- Statistical analysis --- p.70 / Chapter Chapter 4: --- Results --- p.72 / Chapter 4.1 --- "Cloning of the TPAP1 Promoter Fragments, Gene and cDNA" --- p.72 / Chapter 4.1.1 --- TPAP1 promoter fragment constructs --- p.72 / Chapter 4.1.2 --- TPAP1 cDNA cloning --- p.72 / Chapter 4.1.3 --- TPAP1 gene cloning --- p.72 / Chapter 4.2 --- "Sequence analysis of the TPAP1 promoter, gene, cDNA and predicted amino acid sequence" --- p.76 / Chapter 4.2.1 --- "The DNA sequence of the TPAP1 promoter, gene and cDNA" --- p.76 / Chapter 4.2.2 --- Properties of TPAP1 cDNA and protein --- p.83 / Chapter 4.2.3 --- Identification of potential metal ligating residues on TPAP1 --- p.85 / Chapter 4.2.4 --- Phylogenetic relationship of TPAPl to other plant PAPs --- p.86 / Chapter 4.2.5 --- Sequence comparison of 5'UTR ofTPAPl and NtPAP12 --- p.89 / Chapter 4.3 --- APase Activity Assay --- p.90 / Chapter 4.3.1 --- p-NPP APase activity assay --- p.90 / Chapter 4.3.2 --- Activity-gel detection --- p.90 / Chapter 4.4 --- "Comparison of TPAP 1, IAP，SAP 1 and SAP2" --- p.96 / Chapter 4.5 --- Potential Cis-acting Regulatory Elements (CAREs) on the TPAP1 Promoter --- p.100 / Chapter 4.5.1 --- Search for potential CAREs --- p.100 / Chapter 4.5.2 --- Functions of CAREs --- p.100 / Chapter 4.6 --- Transient Expression Analysis --- p.102 / Chapter 4.6.1 --- Biolistic bombardment of TPAP1 promoter fragments into tomato roots --- p.102 / Chapter 4.7 --- Transgenic Expression Analysis --- p.104 / Chapter 4.7.1 --- Transformation of tobacco --- p.104 / Chapter 4.7.2 --- Northern and RT-PCR analysis of GUS expression --- p.110 / Chapter 4.7.3 --- GUS activity analysis --- p.114 / Chapter 4.7.4 --- Histochemical staining of GUS --- p.123 / Chapter Chapter 5: --- Discussions --- p.135 / Chapter 5.1 --- Properties ofTPAPl --- p.135 / Chapter 5.1.1 --- "Structure of the TPAP1 promoter, gene and cDNA" --- p.135 / Chapter 5.1.2 --- Potential flmction(s) ofTPAPl --- p.135 / Chapter 5.1.3 --- The potential relationship between TPAP1 and NtPAP12 --- p.137 / Chapter 5.2 --- Induction of Secretory APases during Pi Starvation --- p.137 / Chapter 5.3 --- Putative Protein Encode by theTPAP 1 cDNA --- p.138 / Chapter 5.4 --- Promoter Analysis of TPAP1 --- p.140 / Chapter 5.4.1 --- Construct preparation --- p.140 / Chapter 5.4.2 --- Potential CAREs located on the TPAP1 promoter --- p.141 / Chapter 5.4.3 --- Transient expression analysis --- p.142 / Chapter 5.4.4 --- Transgenic expression analysis --- p.143 / Chapter 5.4.4.1 --- Northern analysis and RT-PCR analysis of GUS expression --- p.143 / Chapter 5.4.4.2 --- GUS activity analysis --- p.143 / Chapter 5.4.4.3 --- Histochemical staining of GUS --- p.145 / Chapter 5.5 --- Hypothetical Model for TPAP1 Promoter Activities --- p.146 / Chapter 5.5.1 --- Model for expression level --- p.146 / Chapter 5.5.2 --- Models for spatial expressions --- p.148 / Chapter 5.6 --- Future Perspectives --- p.150 / Chapter Chapter 6: --- Conclusions --- p.152 / References --- p.154

Lycopersicon esculentum Mill. cv. Rodade was developed in South Africa for the fresh produce market. This cultivar is also of major importance for South African tomato breeding programmes because of its resistance to bacterial wilt. In this study, aspects of the effects of 6-benzyladenine on adventitious shoot formation by both L. esculentum cv. Rodade and Lycopersicon peruvianum Mill. were studied in vitro. These included the regeneration of adventitious shoots, the effects of different incubation times, the uptake and metabolism of BA and the effect of auxin on the metabolism of BA in both leaf and callus tissue of the two species. Adventitious buds could be regenerated on all tissue types except for callus tissue of L. esculentum. A stepwise increase in the percentage shoots produced was observed indicating a period of induction wherein incubation on a medium containing BA is beneficial to the production of shoots. Leaf tissue was more responsive to BA treatments than callus tissue of both species. The main route of BA metabolism in both species is from BA to [9R]BA and [9R-MP]BA. Callus tissue of L. esculentum cv. Rodade however converted BA to the 3- and 9- glucosides of BA rather than to metabolically active forms of the cytokinin. The auxin, indole-3-acetic acid, played a definite role in the conversion of BA to [3G]BA and [9G]BA in leaf tissue of the tomato cultivar tested, but had no effect in callus tissue of this species. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.