by To Siu-wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 182-195). / ACKNOWLEDGEMENTS --- p.VI / ABSTRACT --- p.VII / LIST OF TABLES --- p.IX / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XVII / Chapter PART I --- GENERAL ASPECTS / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW / Chapter 2.1. --- History of fungal protoplast fusion / Chapter 2.1.1. --- Fungal protoplast preparation technique --- p.4 / Chapter 2.1.2. --- Application of fungal protoplasts --- p.5 / Chapter 2.2. --- Protoplast fusion by polyethene glycol (PEG) --- p.9 / Chapter 2.3. --- Incompatibility system in fungi --- p.10 / Chapter 2.4. --- Characterization of fusion products by genetic markers --- p.12 / Chapter PART II --- OPTIMIZATION OF PROTOPLAST RELEASE AND PROTOPLAST FUSION STUDIES / Chapter CHAPTER 3 --- PROTOPLAST ISOLATION OF Pleurotus florida AND Schizophyllum commune / Chapter 3.1. --- Introduction --- p.14 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Strains and culture media --- p.14 / Chapter 3.2.2. --- Protoplast isolation in different types and concentrations of lytic enzyme --- p.15 / Chapter 3.2.3. --- Protoplast isolation using mycelium with different culture ages --- p.17 / Chapter 3.2.4. --- Protoplast isolation in different types and concentrations of osmotic stabilizers --- p.17 / Chapter 3.2.5. --- Collection of protoplast by centrifugation --- p.18 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Effect of type and concentration of lytic enzyme --- p.19 / Chapter 3.3.2. --- Efficiency of protoplast isolation from mycelia with different culture ages --- p.25 / Chapter 3.3.3. --- Effect of types and concentrations of osmotic stabilizers --- p.28 / Chapter 3.3.4. --- Collecting efficiency of protoplast by centrifugation --- p.31 / Chapter 3.4. --- Discussion / Chapter 3.4.1. --- Choice of lytic enzyme system and time for enzyme digestion --- p.33 / Chapter 3.4.2. --- Culture age for maximum protoplast yield --- p.34 / Chapter 3.4.3. --- Choice of concentration and type of osmotic stabilizers --- p.35 / Chapter CHAPTER 4 --- PROTOPLAST FUSION OF Pleurotus florida AND Schizophyllum commune / Chapter 4.1. --- Introduction --- p.38 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Protoplast formation and size of protoplasts --- p.39 / Chapter 4.2.2. --- Fluorescent staining of protoplasts' nuclei --- p.39 / Chapter 4.2.3. --- Stability of the genetics markers / Chapter 4.2.3.1. --- Preparation of media for checking the presence of genetics markers --- p.40 / Chapter 4.2.3.2. --- Determining the presence of auxotrophic as well as drug resistance markers --- p.42 / Chapter 4.2.4. --- Regeneration of mycelium from protoplast --- p.42 / Chapter 4.2.5. --- Protoplast fusion and screening of fusion products --- p.45 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Size of protoplasts ofPf67 and Scl7 --- p.48 / Chapter 4.3.2. --- Proportion of protoplasts bearing nucleus --- p.48 / Chapter 4.3.3. --- Protoplast regeneration in regeneration medium / Chapter 4.3.3.1. --- Protoplasts regeneration morphologies --- p.52 / Chapter 4.3.3.2. --- Regeneration frequencies and back mutation frequencies of Pf67 and Scl7 protoplasts --- p.58 / Chapter 4.3.4. --- Effect of PEG fusion treatment on auxotrophic and drug resistance markers of Pf67 and Scl7 --- p.60 / Chapter 4.3.5. --- Fusion products obtained from screening process --- p.61 / Chapter 4.4. --- Discussion / Chapter 4.4.1. --- Effect of protoplast isolation and PEG treatment on the two fusion parents --- p.63 / Chapter 4.4.2. --- Structural heterogeneity of protoplasts --- p.64 / Chapter 4.4.3. --- Polymorphic nature of protoplast regeneration --- p.67 / Chapter 4.4.4. --- Protoplast fusion frequence --- p.67 / Chapter PART III --- ANALYSIS OF FUSION PARENTS AND FUSION PRODUCTS / Chapter CHAPTER 5 --- MORPHOLOGICAL AND CYTOLOGICAL STUDIES / Chapter 5.1. --- Introduction --- p.69 / Chapter 5.2. --- Materials and methods / Chapter 5.2.1. --- Strains --- p.69 / Chapter 5.2.2. --- Study on colonial and mycelial morphology --- p.70 / Chapter 5.2.3. --- Fluorescent staining of mycelial nuclei with DAPI --- p.70 / Chapter 5.2.4. --- Study on fruit body and basidial morphology / Chapter 5.2.4.1. --- Fruiting on agar plate --- p.71 / Chapter 5.2.4.2. --- Scanning electron microscopic examination --- p.73 / Chapter 5.3. --- Results / Chapter 5.3.1. --- Variation of colonial morphology --- p.74 / Chapter 5.3.2. --- Morphologies and the number of nuclei in the mycelial cells of fusion parents and fusion products --- p.76 / Chapter 5.3.3. --- Fruit body morphology --- p.82 / Chapter 5.3.4. --- Basidial morphology --- p.84 / Chapter 5.4. --- Discussion --- p.87 / Chapter CHAPTER 6 --- PHYSIOLOGICAL STUDIES OF FUSION PARENTS AS WELL AS FUSION PRODUCTS BY INVESTIGATING THE GROWTH RESPONSES TO DRUGS / Chapter 6.1. --- Introduction --- p.90 / Chapter 6.2. --- Materials and methods / Chapter 6.2.1. --- Strains and media --- p.96 / Chapter 6.2.2. --- Growth responses of the strains to different concentrations of drugs --- p.97 / Chapter 6.3. --- Results / Chapter 6.3.1. --- Comparison of growth pattern as well as growth rate between fusion parents and fusion regenerants --- p.98 / Chapter 6.3.2. --- Growth responses of fusion parents and fusion products on complete medium --- p.105 / Chapter 6.3.3. --- Growth responses of fusion parents and fusion regenerants on complete medium with acriflavin --- p.108 / Chapter 6.3.4. --- Growth responses of fusion parents and fusion products on complete medium with guaiacol --- p.111 / Chapter 6.4. --- Discussion / Chapter 6.4.1. --- General considerations on experimental design --- p.115 / Chapter 6.4.2. --- Growth responses of protoplast regenerants of either fusion parents --- p.116 / Chapter 6.4.3. --- Growth responses on complete medium without fungitoxic drug --- p.117 / Chapter 6.4.4. --- Growth responses on the acriflavin agar medium --- p.118 / Chapter 6.4.5. --- Growth responses on guaiacol agar medium --- p.119 / Chapter 6.4.6. --- Summary --- p.120 / Chapter CHAPTER 7 --- GENETICAL STUDIES / Chapter 7.1. --- Introduction --- p.121 / Chapter 7.2. --- Materials and methods / Chapter 7.2.1. --- Segregation tests of auxotrophic and drug resistance markers in progeny of dikaryotic fusion product --- p.127 / Chapter 7.2.2. --- Complementation test of fusion products as well as the spore germinants of dikaryotic fusion product PS1 --- p.129 / Chapter 7.2.3. --- Recovery of the individual nuclear type of dikaryotic fusion product PS1 --- p.130 / Chapter 7.2.4. --- Genomic fingerprinting / Chapter 7.2.4.1. --- Strains and culture medium --- p.133 / Chapter 7.2.4.2. --- Genomic DNA preparation by cesium chloride (CsCl) method --- p.135 / Chapter 7.2.4.3. --- Genomic DNA preparation by chloroform :TE saturated phenol method --- p.136 / Chapter 7.2.4.4. --- Qualitative analysis of genomic DNA --- p.137 / Chapter 7.2.4.5. --- Quantitative analysis of genomic DNA --- p.137 / Chapter 7.2.4.6. --- DNA amplification by arbitrarily primed -polymerase chain reaction --- p.138 / Chapter 7.3. --- Results / Chapter 7.3.1. --- Progeny analysis and determination of auxotrophic as well as drug resistance markers --- p.140 / Chapter 7.3.2. --- Complementation tests of the fusion products as well as the spore germinants of dikaryotic fusion product PS1 --- p.143 / Chapter 7.3.3. --- Monokaryotic protoplast regenerants of dikaryotic fusion product PS1 --- p.147 / Chapter 7.3.4. --- Studies on extraction of undigested genomic DNA --- p.148 / Chapter 7.3.5. --- Genomic fingerprinting by AP-PCR --- p.155 / Chapter 7.4. --- Discussion / Chapter 7.4.1. --- Genomic DNA extraction --- p.161 / Chapter 7.4.2. --- Recovery of the individual nuclear type of dikaryotic fusion product PS1 --- p.165 / Chapter 7.4.3. --- Genomic changes in fusion products --- p.167 / Chapter 7.4.4. --- Progeny analysis and determination of auxotrophic as well as drug resistance markers --- p.171 / Chapter PART IV --- SUMMING-UP / Chapter CHAPTER 8 --- GENERAL SUMMARY AND CONCLUSION REMARKS / Chapter 8.1. --- General summary --- p.176 / Chapter 8.2. --- Conclusion remarks and future studies --- p.179 / REFERENCES --- p.182 / APPENDIX A SOLUTIONS