Wong Wai Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 175-192). / Abstracts in English and Chinese. / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Abbreviation --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Section I --- The Making of Genetically Modified Organisms --- p.2 / Chapter 1.1 --- Conventional breeding in agriculture --- p.2 / Chapter 1.2 --- What is genetic engineering? --- p.4 / Chapter 1.3 --- Plant transformation --- p.5 / Chapter 1.3.1 --- Agrobacterium-mediated --- p.6 / Chapter 1.3.2 --- Direct gene transfer --- p.8 / Chapter 1.3.2.1 --- Microparticle bombardment --- p.8 / Chapter 1.3.2.2 --- Protoplasts --- p.9 / Chapter 1.3.3 --- Gene silencing --- p.10 / Chapter 1.4 --- Examples of genetically modified crops --- p.13 / Chapter 1.5 --- Foreign genes commonly found in transgenic plants --- p.14 / Chapter Section II --- Benefits and Environmental Concern of GMOs --- p.17 / Chapter 2.1 --- Mechanism of GMO --- p.17 / Chapter 2.1.1 --- Herbicide tolerant crops --- p.18 / Chapter 2.1.2 --- Insect resistant crops --- p.19 / Chapter 2.1.3 --- Delayed ripening crops --- p.20 / Chapter 2.1.4 --- Virus resistant crops --- p.20 / Chapter 2.2 --- Benefits of GMOs --- p.21 / Chapter 2.3 --- Impact of GM foods to human health and the environment --- p.22 / Chapter 2.3.1 --- Human health --- p.22 / Chapter 2.3.1.1 --- GM potatoes --- p.23 / Chapter 2.3.1.2 --- CaMV risks? --- p.24 / Chapter 2.3.1.3 --- Food allergy --- p.25 / Chapter 2.3.2 --- Environmental concerns --- p.26 / Chapter 2.3.2.1 --- Horizontal gene transfer --- p.27 / Chapter 2.3.2.1.1 --- Selectable marker genes --- p.27 / Chapter 2.3.2.1.2 --- Herbicide resistant genes --- p.29 / Chapter 2.3.2.1.3 --- Insect resistant genes --- p.29 / Chapter 2.3.2.2 --- Ecology --- p.30 / Chapter 2.3.2.2.1 --- Monarch butterfly --- p.30 / Chapter Section III --- Future developments of GMO --- p.32 / Chapter 3.1 --- Designer Food and engineered plants --- p.32 / Chapter 3.1.1 --- Insect resistance --- p.33 / Chapter 3.1.2 --- Viral resistance --- p.33 / Chapter 3.1.3 --- Fungal resistance --- p.34 / Chapter 3.1.4 --- Nutritional quality --- p.34 / Chapter 3.1.5 --- Modifications of oil composition --- p.35 / Chapter 3.1.6 --- Medical applications --- p.37 / Chapter 3.1.7 --- Environmental applications --- p.40 / Chapter 3.1.7.1 --- Tolerance to high salinity and drought --- p.40 / Chapter 3.1.7.2 --- Tolerance to frost --- p.41 / Chapter 3.1.7.3 --- Bioremediation --- p.42 / Chapter 3.1.7.4 --- Biodegradable products --- p.43 / Chapter Section IV --- Regulation of GMO --- p.44 / Chapter 4.1 --- The question of labeling --- p.44 / Chapter 4.1.1 --- Moral and ethical issues --- p.44 / Chapter 4.1.2 --- Animal welfare --- p.45 / Chapter 4.2 --- International practice in GMO labeling --- p.46 / Chapter 4.2.1 --- United States of America --- p.46 / Chapter 4.2.2 --- Canada --- p.48 / Chapter 4.2.3 --- European Union --- p.49 / Chapter 4.2.4 --- Australia and New Zealand --- p.50 / Chapter 4.2.5 --- Japan --- p.51 / Chapter 4.2.6 --- Republic of Korea --- p.52 / Chapter 4.2.7 --- China --- p.53 / Chapter 4.2.8 --- Taiwan --- p.53 / Chapter 4.2.9 --- Hong Kong --- p.54 / Chapter Section V --- Uses of crops --- p.56 / Chapter 5.1 --- Uses of crops --- p.56 / Chapter 5.1.1 --- Soybean --- p.56 / Chapter 5.1.2 --- Corn --- p.57 / Chapter 5.1.3 --- Tomato --- p.58 / Chapter 5.1.4 --- Potato --- p.59 / Chapter 5.1.5 --- Rice --- p.60 / Chapter 5.1.6 --- Rapeseed --- p.61 / Chapter 5.1.7 --- Oil --- p.62 / Chapter 5.2 --- "Food additives, hormones and flavourings" --- p.63 / Chapter Chapter 2 --- Materials & Methods --- p.65 / Chapter 2.1 --- Materials --- p.66 / Chapter 2.1.1 --- Growth media & agar --- p.66 / Chapter 2.1.2 --- Reagents for agarose gel electrophoresis --- p.67 / Chapter 2.1.3 --- Reagents for preparation of competent cells --- p.67 / Chapter 2.1.4 --- Reagents for measurement of DNA concentration --- p.68 / Chapter 2.1.4.1 --- Measurement of DNA concentration by PicoGreen --- p.68 / Chapter 2.1.5 --- Reagents for Southern hybridization --- p.68 / Chapter 2.2 --- Methods --- p.70 / Chapter 2.2.1 --- Restriction endonuclease digestion --- p.70 / Chapter 2.2.2 --- Agarose gel electrophoresis of DNA --- p.70 / Chapter 2.2.3 --- DNA recovery from agarose gel --- p.71 / Chapter 2.2.3.1 --- QIAquick® gel extraction --- p.71 / Chapter 2.2.4 --- Ligation of purified DNA fragment into vector --- p.72 / Chapter 2.2.5 --- Transformation --- p.72 / Chapter 2.2.6 --- Rubidium chloride method for making competent cells --- p.12 / Chapter 2.2.7 --- Plasmid DNA preparation --- p.73 / Chapter 2.2.7.1 --- Concert Rapid Mini Prep --- p.73 / Chapter 2.2.7.2 --- QIAprep® Miniprep --- p.74 / Chapter 2.2.8 --- Extraction of plant genomic DNA --- p.75 / Chapter 2.2.8.1 --- Qiagen DNeasy´ёØ Plant Mini Kit --- p.75 / Chapter 2.2.9 --- Southern Hybridization --- p.75 / Chapter 2.2.9.1 --- Denaturation --- p.76 / Chapter 2.2.9.2 --- Blot transfer --- p.76 / Chapter 2.2.9.3 --- Pre-hybridization --- p.77 / Chapter 2.2.9.4 --- Synthesis of radiolabelled probe --- p.77 / Chapter 2.2.9.5 --- Hybridization of radiolabelled probe on filter --- p.77 / Chapter 2.2.9.6. --- Detection of hybridized probes --- p.78 / Chapter 2.2.10 --- Measurement of DNA concentration --- p.78 / Chapter 2.2.10.1 --- Determination of DNA on EtBr stained gel --- p.78 / Chapter 2.2.10.2 --- Determination of DNA by UV spectrophotometer --- p.78 / Chapter 2.2.10.3 --- Determination of DNA by PicoGreen --- p.79 / Chapter 2.2.11 --- DNA sequencing --- p.80 / Chapter 2.2.11.1 --- Automated sequencing by ABI Prism 377 --- p.80 / Chapter Chapter 3 --- PCR Diagnostics --- p.81 / Chapter 3.1 --- Applications of PCR to processed foods --- p.82 / Chapter 3.1.1 --- DNA quality --- p.82 / Chapter 3.1.2 --- PCR & Multiplex PCR --- p.83 / Chapter 3.1.3 --- Choice of primers --- p.84 / Chapter 3.1.4 --- Inhibitors --- p.84 / Chapter 3.2 --- Materials & Methods --- p.85 / Chapter 3.2.1 --- Selection of primers --- p.85 / Chapter 3.2.2 --- Amplification of target sequences --- p.86 / Chapter 3.2.3 --- Multiple amplification of target sequences --- p.87 / Chapter 3.3 --- Results --- p.88 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Quality Control in GMO detection --- p.95 / Chapter 4.1 --- Standardization of pre- and post- PCR analysis --- p.96 / Chapter 4.1.1 --- General guidelines --- p.96 / Chapter 4.1.2 --- UV irradiation --- p.97 / Chapter 4.1.3 --- Inactivation protocols --- p.93 / Chapter 4.1.4 --- Positive and negative controls --- p.99 / Chapter 4.1.5 --- PCR verification --- p.99 / Chapter 4.1.6 --- Equipment decontamination --- p.100 / Chapter 4.2 --- Materials & Methods --- p.101 / Chapter 4.2.1 --- Selection of primers for external control --- p.101 / Chapter 4.2.2 --- Development of the external control --- p.101 / Chapter 4.2.3 --- Selection of primers for internal control --- p.103 / Chapter 4.3 --- Results --- p.104 / Chapter 4.4 --- Discussion --- p.107 / Chapter Chapter 5 --- DNA extraction from food samples --- p.110 / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.2 --- Reagents and Buffers for DNA extraction from food samples --- p.112 / Chapter 5.2.1 --- Cetyltrimethylammonium bromide (CTAB) extraction method --- p.112 / Chapter 5.2.2 --- Organic-based extraction method --- p.113 / Chapter 5.2.3 --- Potassium acetate/sodium dodecyl sulphate precipitation method --- p.113 / Chapter 5.2.4 --- Hexane-based extraction method --- p.114 / Chapter 5.3 --- Weight and names of samples --- p.115 / Chapter 5.4 --- DNA extraction methods --- p.115 / Chapter 5.4.1 --- CTAB extraction method --- p.115 / Chapter 5.4.2 --- Qiagen DNeasy´ёØ plant mini kit --- p.116 / Chapter 5.4.3 --- Promega Wizard® genomic DNA purification --- p.116 / Chapter 5.4.4 --- Promega Wizard® Magnetic DNA purification system --- p.117 / Chapter 5.4.5 --- Promega Wizard® DNA Clean-Up system --- p.118 / Chapter 5.4.6 --- Qiagen QIAshreddrer´ёØ and QIAamp spin column --- p.119 / Chapter 5.4.7 --- Chelex-based extraction method --- p.119 / Chapter 5.4.8 --- Organic-based extraction method --- p.120 / Chapter 5.4.9 --- Nucleon PhytoPure extraction and purification method --- p.120 / Chapter 5.4.10 --- Potassium acetate/SDS precipitation method --- p.121 / Chapter 5.4.11 --- Hexane-based extraction method --- p.122 / Chapter 5.5 --- Results --- p.123 / Chapter 5.5.1 --- Comparison of eleven extraction methods --- p.123 / Chapter 5.5.2 --- Comparison of DNA extraction on selected methods --- p.125 / Chapter 5.6 --- Discussion --- p.132 / Chapter Chapter 6 --- Quantitative Analysis --- p.136 / Chapter 6.1 --- Introduction --- p.137 / Chapter 6.1.1 --- Chemistry of quantitative PCR --- p.138 / Chapter 6.1.2 --- PCR system --- p.140 / Chapter 6.2 --- Materials & Methods --- p.142 / Chapter 6.2.1 --- Design of primers and probes --- p.142 / Chapter 6.2.2 --- Methods --- p.145 / Chapter 6.3 --- Results --- p.146 / Chapter 6.3.1 --- Selection of primer/probe --- p.146 / Chapter 6.3.2 --- Primer optimization --- p.149 / Chapter 6.3.3 --- Quantitative analysis of real samples --- p.158 / Chapter 6.4 --- Discussion --- p.152 / Chapter Chapter 7 --- Conclusion --- p.168 / References --- p.175 / Appendix --- p.193

Genetic improvement of the potato through classical breeding has been limited by its tetraploid nature, the narrow genetic variability within cultivars, and interploidy barriers between tetraploid cultivars and diploid germplasm. Breeding at reduced ploidy levels has been proposed as a solution to these problems. Because of sterilities, somatic hybridization via protoplast fusion has been considered an alternative to sexual polyploidization for resynthesizing superior diploids from selected monoploids, and tetraploids from selected diploids and dihaploids. Successful application of somatic hybridization largely depends upon protoplast culturability and regenerability of a plant. The ability of callus formation and plant regeneration from protoplasts varies among plants. To understand the genetic basis for this variation, the mode of inheritance for protoplast culturability, defined as the ability to develop calli from cultured protoplasts, was studied in the diploid potato species, Solanum phureja. Based upon data from F₂ as well as from F₁ and backcross progenies, it was found that protoplast culturability in this potato species was controlled by two unlinked loci with dominant effect. In addition, there was quantitative variation for protoplast plating efficiency among culturable genotypes. Male sterility in cultivars of Solanum tuberosum ssp. tuberosum results from nuclear-cytoplasmic interactions. 'Donor-recipient' protoplast fusion and regeneration were conducted between a sterile S. tuberosum ssp. tuberosum cultivar, Russet Burbank, and fertile selections of S. tuberosum ssp. andigena which have a non-sensitive cytoplasm and were used as the cytoplasmic donor. Sixteen regenerated plants possessed nuclear background and chloroplast DNA of Russet Burbank. However, two of these regenerants had improved pollen stainability. The possible causes for the improvement of pollen stainability are discussed. In the last chapter, allelic polymorphism in a monoploid population derived from anther culture of a clone of S. phureja was assessed by isozyme electrophoresis. Fourteen monoploids and their anther donor were examined for six enzymes. No allozyme variation was detected in these plants. However, genetic variability among these monoploids was manifested by variations in some growth characters and general morphology. The limitation of enzymatic markers in detecting allelic polymorphism in these monoploids is discussed. / Ph. D.