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Many species from economically important genera remain rooting recalcitrant, prohibiting the commercialisation of many species in forestry and horticulture, and hindering genetic improvement by conventional breeding or recombinant DNA technology, where vegetative propagation is often used to preserve the genetic fidelity of elite progeny. Two cherry species (Prunus avium and P. padus) were used as models in this study to investigate the physiological and genetic manipulation of adventitious rooting. Mature trees are typically more difficult to propagate vegetatively than their juvenile counterparts. For some trees, micropropagation can circumvent certain effects of ageing and maturation, restoring shoot vigour and rooting, but the mechanism(s) involved have not been elucidated. During micropropagation, subculture interval was found not to be the predominant factor promoting the 'apparent rejuvenation' of mature P. avium tissue. 'Apparently rejuvenated' ex vitro and hedged (putatively) mature P. avium trees were treated with gibberellins predicted to have a range of structural related activities. GA, improved the rooting of cuttings from hedged (putatively) mature cherry, but not from ex vitro trees. Methodology to regenerate adventitious shoots from P. avium leaf explants was developed, (putative) transgenic P. padus plants were produced by an Agrobacterium tumefaciens mediated strategy. Auxin redistribution in planta is postulated to require a component of active transport; inhibition of the predominantly basipetal transport has profound effects on rooting. The putative function of the Arabidopsis thaliana AtAUX1 gene is that of a cellular auxin influx carrier, possibly, as described by the chemiosmotic hypothesis. This thesis examined the hypothesis that transformation with the AtAUX1 gene would enhance the delivery of the root-inducing signal to improve rooting of P. padus, a species which is rooting recalcitrant and more or less obligate on exogenous auxin for this process. However, all six, constitutively expressed, Cauliflower Mosaic Virus 35S promoter driven, 35S::AtAUX1, transgenic shoot lines had reduced rooting.

Lu Shanxiang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 122-135). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Lysine-rich protein from winged bean --- p.2 / Chapter 1.1.1 --- Discovery --- p.2 / Chapter 1.1.2 --- Applications in enhancing nutritional values --- p.2 / Chapter 1.2 --- Plant secretory pathway --- p.4 / Chapter 1.2.1 --- Overview of plant secretory pathway --- p.4 / Chapter 1.2.2 --- Three models on protein transportation from ER to Golgi --- p.6 / Chapter 1.2.3 --- Brefeldin A: inhibitor of secretion --- p.9 / Chapter 1.2.4 --- Markers for different organelles --- p.10 / Chapter 1.3 --- Tobacco bright yellow 2 (BY-2) cell system --- p.11 / Chapter 1.3.1 --- Origin of BY-2 cell line --- p.12 / Chapter 1.3.2 --- Characteristics of BY-2 cell line --- p.12 / Chapter 1.4 --- Use of fluorescent proteins as reporters --- p.13 / Chapter 1.4.1 --- GFP and its derivatives --- p.13 / Chapter 1.4.2 --- Reporter system --- p.15 / Chapter 1.4.3 --- Applications of GFP and its derivatives in plants --- p.16 / Chapter 1.5 --- Temperature effects on plants --- p.17 / Chapter 1.6 --- Project objectives --- p.18 / Chapter Chapter 2 --- Subcellular localization of LRP in winged bean (Psophocarpus tetragonolobus)seeds --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Chemicals --- p.21 / Chapter 2.2.2 --- Plant materials --- p.21 / Chapter 2.2.3 --- Antibodies --- p.22 / Chapter 2.2.4 --- Western blot --- p.23 / Chapter 2.2.4.1 --- Protein extraction --- p.23 / Chapter 2.2.4.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.23 / Chapter 2.2.4.3 --- Immunodetection --- p.24 / Chapter 2.2.5 --- Confocal Immunofluorescence --- p.25 / Chapter 2.2.5.1 --- Preparation of samples for immuno-labeling --- p.25 / Chapter 2.2.5.2 --- Immuno-labeling --- p.26 / Chapter 2.2.5.3 --- Collection and analysis of confocal fluorescent images --- p.27 / Chapter 2.2.6 --- Immuno transmission electron microscope (TEM) study --- p.28 / Chapter 2.2.6.1 --- Preparation of samples --- p.28 / Chapter 2.2.6.2 --- Immuno-labeling --- p.29 / Chapter 2.3 --- Results --- p.31 / Chapter 2.3.1 --- Anti-alpha-TIP and anti-LRP antibodies have good specificity in winged bean seeds --- p.31 / Chapter 2.3.2 --- Anti-a-TIP antibodies could label the PSVs of winged bean seeds specifically --- p.31 / Chapter 2.3.3 --- LRP was localized outside of PSVs --- p.33 / Chapter 2.3.4 --- Immuno-TEM localization of LRP --- p.35 / Chapter 2.4 --- Conclusion and discussion --- p.40 / Chapter Chapter 3 --- Generation of Transgenic Tobacco BY-2 Cell Lines Expressing YFP and LRP Fusions --- p.41 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.42 / Chapter 3.2.1 --- Primers --- p.42 / Chapter 3.2.2 --- Plant materials --- p.43 / Chapter 3.2.3 --- Bacterial strains --- p.44 / Chapter 3.2.4 --- Construction of fusion constructs --- p.44 / Chapter 3.2.4.1 --- Four fusion constructs of LRP and YFP --- p.44 / Chapter 3.2.4.2 --- His-tag-YFP fusion construct --- p.45 / Chapter 3.2.4.3 --- Cloning of the fusion protein genes into Agrobacterium binary vector pBI121 --- p.45 / Chapter 3.2.5 --- Confirmation of the fusion constructs --- p.53 / Chapter 3.2.6 --- Transformation of Agrobacterium by electroporation --- p.53 / Chapter 3.2.7 --- "Transformation, selection and suspension of tobacco BY-2 cells" --- p.54 / Chapter 3.2.8 --- "Transformation, screening and induction of E. coli BL21-DE3 for expression of His-tagged YFP" --- p.55 / Chapter 3.2.9 --- Protein extraction --- p.55 / Chapter 3.2.9.1 --- Protein fractionation from BY-2 cells --- p.55 / Chapter 3.2.9.2 --- protein extraction from E. coli of BL21-DE3 --- p.56 / Chapter 3.2.10 --- Immunolabeling of suspension cultured cells --- p.56 / Chapter 3.2.11 --- Raising anti-GFP antibodies --- p.57 / Chapter 3.2.12 --- Dot blot analysis --- p.58 / Chapter 3.2.13 --- Affinity purification of proteins and antibodies --- p.59 / Chapter 3.2.13.1 --- Metal affinity resin column for protein purification --- p.59 / Chapter 3.2.13.2 --- Cyanogens bromide (CNBr) activated sepharose column for antibody purification --- p.60 / Chapter 3.2.14 --- SDS-PAGE and western blot analysis --- p.61 / Chapter 3.2.15 --- Antibodies --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Two transgenic BY-2 cell lines showed different fluorescent signal patterns --- p.62 / Chapter 3.3.2 --- Two cell lines showed different fluorescent signal stability --- p.63 / Chapter 3.3.3 --- The two fusion proteins were localized in different places in the BY-2 cells --- p.67 / Chapter 3.3.4 --- """Green"" E. coli expressed the recombinant YFP" --- p.69 / Chapter 3.3.5 --- Expressed recombinant YFP could not be affinity purified --- p.69 / Chapter 3.3.6 --- Raised polyclonal anti-GFP antibodies showed good specificity --- p.69 / Chapter 3.4 --- Conclusion and discussion --- p.75 / Chapter Chapter 4 --- Characterization of SpYFP-LRP Fusion in Transgenic BY-2 cells --- p.76 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.77 / Chapter 4.2.1 --- Plant materials --- p.77 / Chapter 4.2.2 --- BFA and heat treatment --- p.77 / Chapter 4.2.3 --- Confocal immunolabeling --- p.78 / Chapter 4.2.4 --- Conventional TEM study --- p.78 / Chapter 4.2.5 --- Immuno TEM using Lowicryl resin and LR White resin --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- "BFA induced the SpYFP-LRP-marked organelle to form ""BFA-induced"" compartments" --- p.82 / Chapter 4.3.2 --- Partial recovery from BFA treatment --- p.84 / Chapter 4.3.3 --- SpYFP-LRP was localized in BFA-induced compartments --- p.87 / Chapter 4.3.4 --- BFA treatment induced the formation of various compartmentsin SpYFP-LRP cells --- p.90 / Chapter 4.3.5 --- BFA-induced structures contain SpYFP-LRP --- p.99 / Chapter 4.3.6 --- Elevated temperature affected the signal pattern but not the localization of SpYFP-LRP in transgenic BY-2 cells --- p.100 / Chapter 4.3.7 --- Elevated temperature treatment induced the SPYFP-LRP cells to form new vesicular compartments --- p.105 / Chapter 4.4 --- Conclusions and Discussion --- p.112 / Chapter 4.4.1 --- BFA treatment --- p.112 / Chapter 4.4.2 --- Heat treatment --- p.114 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.116 / Chapter 5.1 --- Summary --- p.117 / Chapter 5.2 --- Future perspectives --- p.119 / Reference --- p.122

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Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

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Thesis (M.S.)--Texas State University--San Marcos, 2009. / Vita. Reproduction permission applies to print copy: Blanket permission granted per author to reproduce. Includes bibliographical references (leaves 68-70).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 146-161).

Thesis (Ph. D.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references (leaves 171-190). Also available on the World Wide Web.