Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

The usefulness of interspecific crosses between Sitka spruce (Picea sitchensis (Bong.) Carr.) and interior spruce (Picea glauca (Moench) Voss, Picea engelmannii Parry and their hybrids) was investigated in one coastal environment. For height growth and bud set most of the variation was at the regional and individual cross level, but very little variation was at the subregional level. The main genetic effects, male and female, accounted for a majority of the genetic variance and additive genetic effects are interpreted as the main factor in the determination of height growth and bud set. For bud set the maternal source of variation accounted for the majority of the genetic variance and a maternal influence on bud set is suggested. Some specific cross combinations were outstanding in height growth and non-additive genetic factors are considered important in these crosses. For bud break most of the variation was due to the residual error, although regions and crosses were statistically significant sources of variation. The Female*Male term was the most important genetic source of variation although bud break is not considered to have as much genetic variation as height and bud set. Large differences were found in the pattern of cold hardiness in the fall and it is considered that photoperiod plays a much larger role than previously thought, especially for interior spruce. Most of the variation was again at the regional and individual cross level. The intermediate performance of the hybrids suggests an inheritance of cold-hardiness based on additive genetic effects. Recommendations are given in the text for the use of these hybrids as well as the areas in which further research would be desireable. / Forestry, Faculty of / Graduate

Call number: LD2668 .T4 1978 K44 / Master of Science

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm. / AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by sukrosemetabolisme, te bepaal. Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes. Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens alle fases van suikkerrietontwikkeling moontlik gemaak het. Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed. RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die suikerrietstingel te bepaal.

Four species of the genus Parthenium were involved in a hybridization attempt. The species were: guayule (Parthenium argentatum Gray), Parthenium fruticosum Less, Parthenium bipinnatifidum (Ortega) Rollins, and Parthenium incanum H. B. K. Primary attention was given to the formation of hybrids between P. argentatum and P. fruticosum. Characteristics of the latter species such as size, growth rate, possible disease resistance, and wider geographical adaptation were desirable for transfer to P. argentatum. Reciprocal crosses were made between these two species and the production of hybrids was not difficult. However, in the case of selfing, backcross and sibcrosses, germination percent was 0.86 from a total of 3,471 achenes. Therefore, a search for the cause or causes of the negative results was initiated. The factors which were considered of primary interest were: incompatibility, genic and chromosomal sterility, pollination and planting techniques, and seed germination treatment. Incompatibility was considered the main limiting factor in the formation of a backcross population. Rubber analysis was performed in the interspecific hybrids. Mean rubber percent for hybrids between P. artentatum and P. fruticosum was 1.19; and for the reciprocal cross it was 0.39. Hybrids of the cross P. fruticosum x P. bipinnatifidum had a mean rubber percent of 0.19.

by Lau Wing Chung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1985 / Bibliography: leaves 209-236

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

by Jiong Zhao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 197-217). / Acknowledgments --- p.III / Abstract --- p.IX / Abbreviations --- p.XI / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- What is a mushroom? --- p.1 / Chapter 1.2 --- Mushroom Genetics: its development and prospective --- p.1 / Chapter 1.2.1 --- Genome karyotype by pulsed field gel electrophoresis analysis --- p.2 / Chapter 1.2.2 --- Mitochondrial Genetics --- p.4 / Chapter 1.2.3 --- Mating type genes --- p.5 / Chapter 1.2.4 --- Transformation --- p.7 / Chapter 1.2.5 --- Parasexual processes --- p.8 / Chapter 1.2.6 --- Mushroom breeding --- p.11 / Chapter Chapter 2. --- Literature review: Protoplast fusion in fungi --- p.14 / Chapter 2.1 --- Introduction --- p.14 / Chapter 2.2 --- Protoplast fusion in yeasts --- p.14 / Chapter 2.2.1 --- Intraspecific fusion --- p.14 / Chapter 2.2.2 --- Interspecific fusion --- p.15 / Chapter 2.2.3 --- Intergeneric fusion --- p.16 / Chapter 2.3 --- Protoplast fusion in some Filamentous fungi --- p.17 / Chapter 2.3.1 --- Aspergillus --- p.17 / Chapter 2.3.2 --- Fusarium --- p.18 / Chapter 2.3.3 --- Tricoderma --- p.19 / Chapter 2.4 --- Protoplast fusion in strains --- p.21 / Chapter 2.4.1 --- Protoplast isolation and regeneration --- p.21 / Chapter 2.4.2 --- Intraspecific fusion in mushroom species --- p.24 / Chapter 2.4.3 --- Interspecific fusion in mushroom species --- p.24 / Chapter 2.4.4 --- Intergeneric fusion in mushroom species --- p.26 / Chapter 2.4.5 --- Transfer of nuclei in mushroom species --- p.27 / Chapter 2.5 --- General conclusions about literatures --- p.27 / Chapter 2.5.1 --- Brief points about fungal protoplast fusion --- p.27 / Chapter 2.5.2 --- Some arguements about fusion works in mushrooms strains --- p.31 / Chapter 2.5.2.1 --- Classification of parental strains --- p.31 / Chapter 2.5.2.2 --- Control experiments --- p.31 / Chapter 2.5.2.3 --- Indentification methods of hybrids --- p.32 / Chapter 2.6 --- General research ideas about experiments --- p.33 / Chapter Chapter 3 --- Protoplast isolation and regeneration in some mushroom species --- p.37 / Chapter 3.1 --- Introduction --- p.37 / Chapter 3.2 --- Materials and Methods --- p.38 / Chapter 3.2.1 --- Strains --- p.38 / Chapter 3.2.2 --- Media --- p.38 / Chapter 3.2.3 --- Protoplast release --- p.40 / Chapter 3.2.4 --- Protoplast regeneration --- p.41 / Chapter 3.3 --- Results and Discussion --- p.41 / Chapter 3.3.1 --- Effect of culture age --- p.41 / Chapter 3.3.2 --- Effect of lytic enzyme --- p.42 / Chapter 3.3.3 --- Effect of concentration of mycelium --- p.45 / Chapter 3.3.4 --- Effect of filter system --- p.46 / Chapter 3.3.5 --- Effect of different regeneration protocols --- p.48 / Chapter 3.3.6 --- Effect of soluable starch --- p.49 / Chapter 3.3.7 --- Effect of PEG on the regeneration frequency --- p.50 / Chapter 3.4 --- Conclusions --- p.53 / Chapter Chapter 4 --- Monokaryotization by protoplasting technique in some heterothallic mushroom species --- p.54 / Chapter 4.1 --- Introduction --- p.54 / Chapter 4.2 --- Materials and Methods --- p.55 / Chapter 4.2.1 --- Strains and media --- p.55 / Chapter 4.2.2 --- Production of neo-monokaryons by protoplast technique --- p.55 / Chapter 4.2.3 --- Identification of mating types in protoplasted monokaryons --- p.57 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Formation of neo-monokaryons --- p.57 / Chapter 4.3.2 --- Monokaryotization in different strains --- p.60 / Chapter 4.3.3 --- Comparison of parental and protoplasted monokaryons --- p.60 / Chapter 4.3.4 --- Comparison of regeneration rate of parental monokaryons --- p.62 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Differences of regeneration time in monokaryons and dikaryons --- p.64 / Chapter 4.4.2 --- Genetic differences between parental and neo-monokaryons --- p.64 / Chapter 4.4.3 --- Mechanism for the production of neo-monokaryons --- p.65 / Chapter 4.4.4 --- Advantages of protoplasting technique in mushroom breeding --- p.65 / Chapter 4.4.5 --- Protoplasting technique in the identification of fusion hybrids --- p.67 / Chapter 4.5 --- Couclusions --- p.68 / Chapter Chapter 5 --- Intraspecific hybridization in Coprinus cinereus and Schizophyllum commune by PEG-induced protoplast fusion and electrofusion --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.2 --- Materials and Methods / Chapter 5.2.1 --- Strains and Media --- p.70 / Chapter 5.2.2 --- Fusogen --- p.70 / Chapter 5.2.3 --- Inactivation chemicals --- p.71 / Chapter 5.2.4 --- Inactivation of protoplasts --- p.71 / Chapter 5.2.5 --- PEG induced protoplast fusion --- p.72 / Chapter 5.2.6 --- Electrofusion --- p.72 / Chapter 5.2.7 --- Investigation of protoplast fusion yield and fusion frequency --- p.73 / Chapter 5.2.8 --- Comparison of mycelium growth rate --- p.73 / Chapter 5.2.9 --- Fruiting test --- p.74 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Inactivation by IA and DP --- p.76 / Chapter 5.3.2 --- Effect of different fusogens on fusion frequency --- p.79 / Chapter 5.3.3 --- Effect of different fusion protocols on fusion frequency --- p.79 / Chapter 5.3.4 --- Optimization of electrofusion --- p.80 / Chapter 5.3.5 --- Fusion frequency resulted by PEG and electrofusion --- p.83 / Chapter 5.3.6 --- Comparison of colony diameters and fruiting time --- p.84 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- Inactivation of protoplasts by biochemical inhibitors --- p.85 / Chapter 5.4.2 --- Optimization of PEG induced fusion --- p.86 / Chapter 5.4.3 --- Optimization of electrofusion --- p.86 / Chapter 5.4.4 --- Identification of fusion heterokaryons --- p.87 / Chapter 5.4.5 --- Comparison of PEG and electrofusion --- p.89 / Chapter 5.4.2 --- Effect of mitochondria --- p.90 / Chapter 5.5 --- Couclusions --- p.91 / Chapter Chapter 6 --- Interspecific hybridization between Volvariella volvacea and Volvariella bomhycina by protoplast fusion --- p.92 / Chapter 6.1 --- Introduction --- p.92 / Chapter 6.2 --- Materials and Methods / Chapter 6.2.1 --- Strains and Media --- p.93 / Chapter 6.2.2 --- Protoplast production and regeneration --- p.94 / Chapter 6.2.3 --- Inactivation of protoplasts --- p.94 / Chapter 6.2.4 --- Protoplast fusion --- p.94 / Chapter 6.2.5 --- Selection of fusion products --- p.95 / Chapter 6.2.6 --- Analyses of progeny --- p.95 / Chapter 6.2.7 --- Identification of fusants by protoplasting technique --- p.96 / Chapter 6.2.8 --- Nuclear DNA contents in parents and hybrids --- p.96 / Chapter 6.2.9 --- Genomic DNA amplification by arbitraly primers --- p.96 / Chapter 6.2.10 --- Amplification by nuclear and mitochondrial rDNA --- p.97 / Chapter 6.2.11 --- Fruiting test --- p.97 / Chapter 6.3 --- Results / Chapter 6.3.1 --- Inactivation of Vb10 protoplasts --- p.98 / Chapter 6.3.2 --- Low temperature effect on Vv34 --- p.100 / Chapter 6.3.3 --- Selection of fusants --- p.100 / Chapter 6.3.4 --- Analyses of progeny --- p.106 / Chapter 6.3.5 --- Identification by protoplasting technique --- p.108 / Chapter 6.3.6 --- Nuclear DNA contents in parents and hybrids --- p.110 / Chapter 6.3.7 --- Arbitraly primer amplified PCR fingerprinting --- p.113 / Chapter 6.3.8 --- rDNA PCR results --- p.119 / Chapter 6.3.9 --- Interspecific variations / Chapter 6.3.10 --- Genome analysis of hybrids by pulse field gel electrophoresis / Chapter 6.3.11 --- Fruiting test / Chapter 6.4 --- Discussion / Chapter 6.4.1 --- Strain choice --- p.125 / Chapter 6.4.2 --- Low temperature strains --- p.125 / Chapter 6.4.3 --- Nuclear DNA content --- p.125 / Chapter 6.4.4 --- AP-PCR and RAPDs markers --- p.126 / Chapter 6.4.5 --- Interspecific fusion in Volvariella --- p.126 / Chapter 6.5 --- Couclusions --- p.130 / Chapter Chapter 7 --- Intergeneric hybridization between Schizophyllum commune and Pleurotus florida by protoplast fusion --- p.131 / Chapter 7.1 --- Introduction --- p.131 / Chapter 7.2 --- Materials and Methods / Chapter 7.2.1 --- Strains and Media --- p.132 / Chapter 7.2.2 --- Protoplast fusion --- p.133 / Chapter 7.2.3 --- Analyses of progeny --- p.134 / Chapter 7.2.4 --- Phylogenetic analysis --- p.135 / Chapter 7.2.5 --- Fruiting test --- p.135 / Chapter 7.3 --- Results / Chapter 7.3.1 --- Selection of fusion products --- p.135 / Chapter 7.3.2 --- Analyses of fusion progeny --- p.139 / Chapter 7.3.3 --- Identification by protoplasting technique --- p.143 / Chapter 7.3.4 --- Determination of nuclear DNA contents --- p.145 / Chapter 7.3.5 --- rDNA PCR analysis in fusion --- p.148 / Chapter 7.3.6 --- Identification of hybrids by AP-PCR and RAPDs markers --- p.151 / Chapter 7.3.7 --- Phylogenetic analysis --- p.162 / Chapter 7.3.8 --- Fruiting test --- p.164 / Chapter 7.4 --- Discussion --- p.165 / Chapter 7.5 --- Couclusions --- p.169 / Chapter Chapter 8 --- Protoplast fusion in shiitake and other species --- p.171 / Chapter 8.1 --- Introduction --- p.172 / Chapter 8.2 --- Materials and Methods --- p.172 / Chapter 8.3 --- Results and Discussion --- p.173 / Chapter 8.4 --- Couclusion --- p.179 / Chapter Chapter 9. --- General discussion and conclusions --- p.180 / Appendix 1. Determination of ploidy in some mushrooms --- p.187 / Appendix 2. Genomic DNA Isolation --- p.188 / Appendix 3. Arbitrary primer polymerase chain reaction --- p.190 / Appendix 4. rDNA PCR Amplification conditions --- p.193 / Appendix 5. Pulsed Field Gel Electrophoresis --- p.195 / Appendix 6. Genetic distance analysis in hybrids and their parents --- p.196 / References --- p.197

雜交在植物育種中起到重要的作用，但是，雜交障礙阻礙基因資源在遠源物種間流動而未能使其應用於植物育種中。雜交障礙基本上分為配子融合前障礙和配子融合後障礙。離體雜交技術可以用來克服配子融合前障礙，如空間和時間的隔離、花期的分離、花粉和雌蕊的不親和性。配子融合後障礙比較複雜，如雜種生存能力差、活力弱和不育，染色體加倍、配拯救和體細胞雜交技術可以用來應對這些障礙。但是，這些技術不能解決染色體消除障礙。染色體消除現象可以在遠源物種間的配子體雜種和體細胞雜種中觀察到，表現為在胚的生長發育過程中一個親本的染色體組隨著有絲分裂的進程完全失，這種現象可以在小麥和玉米的雜種中觀察到。為了克服這種雜交障礙，我們假定在小麥中過量表達玉米的CenH3基因可以促進玉米染色體在小麥中保留，進而克服這種雜交障礙。這種策略如果起作用的話，可以應用到創造遠源雜種，轉移染色體，尤其是轉移迷你染色體並應用於作物的遺傳改良。 / 在本研究中，應用小麥和玉米的雜交系統來驗證CenH3基因是否可以解決染色體消除障礙。通過基因槍介導基因轉化的方法，兩個載體分別被轉化到小麥幼胚中，這兩個載體分別含有YFP標記的玉米CenH3基因，和RFP標記的融合CenH3基因用來表達含有小麥CENH3蛋白loop 1之前以及玉米CENH3蛋白loop 1之後的融合蛋白。通過PCR、RFP熒光標記和熒光原位雜交的方法鑑定了含有融合CENH3蛋白的轉基因小麥，並且這些轉基因小麥是可育和穩定遺傳的植株。但是并没有得到含有玉米CenH3基因的轉基因小麥。因此，含有融合CENH3蛋白的轉基因小麥作為受體應用於同玉米的雜交中。 / 通過體細胞雜交的方法來構建轉基因小麥和玉米的體細胞雜種，並研究該雜種細胞中染色體的行為。通過醋酸地衣紅染色的方法對不同時期含有融合CENH3蛋白小麥和玉米的融合細胞的分析表明該融合CENH3蛋白不能完全消除有絲分裂過程中異常染色體行為的現象。熒光原位雜交分析同樣表明該融合CENH3蛋白可能未能阻止雜種中染色體消除的現象，但是需要更多的結果去驗證該融合CENH3蛋白作用並得出最終結論。 / Hybridization plays an important role in plant breeding, but hybridization barriers block the way of gene flow to use far related genetic resources for breeding. Hybridization barriers are generally classified into pre-zygotic and post-zygotic barriers. Pre-zygotic barriers such as spatial and temporal separation, floral isolation, and pollen-pistil incompatible can be overcome by in vitro hybridization technology. Post-zygotic barriers such as hybrid non-viability, weakness, and sterility are more complicated, and techniques such as chromosome doubling, embryo rescue, and somatic hybridization have been developed to tackle with these problems. However, chromosome eliminations can not be solved by these techniques. Chromosome eliminations are observed in zygotes and somatic hybrids of far related plant species, where chromosomes of one genome can be completely lost in mitosis during the embryo development. Such phenomena were observed in wheat x maize hybridizations. To overcome this kind of barrier, we hypothesize that over expression of maize CenH3 gene in wheat could facilitate maize chromosome adaptation in the hybrids, and thus overcome the hybridization barrier. This strategy, if it works, could be applied to make wide crosses, and transfer chromosomes, especially minichromosome for crop improvements. / In this study, wheat x maize intergenic hybridization system was used to test whether CenH3 gene could be used to solve chromosome eliminations. Two constructs that contained a YFP tagged maize CenH3 gene, and a RFP tagged fusion CenH3 gene of wheat CENH3 before loop 1 and maize CENH3 after loop 1, were delivered to wheat immature embryos respectively by gold particle bombardment approach. Fertile and stable transgenic wheat plants with fusion CENH3 (fusion CENH3 wheat) were produced, and confirmed by PCR, RFP fluorescence detection, and FISH analysis, while transgenic wheat plants with maize CenH3 gene were not obtained. Transgenic wheat plants that expressed the fusion CenH3 gene were used as recipients in hybridizations with maize. / Somatic hybridization between transgenic wheat and maize were performed to investigate chromosome behaviors in the hybrids. Aceto-orcein staining analysis of fusion protoplast of fusion CENH3 wheat and maize at different stages showed that the fusion CENH3 could not completely eliminate abnormal chromosome behaviors during mitosis. FISH analysis also suggested that fusion CENH3 might not prevent chromosome elimination in the hybrids, but more results are needed to make final conclusions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Jianhui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 90-97). / Abstracts also in Chinese. / List of Figures --- p.iii / List of Tables --- p.iv / List of Abbreviations --- p.v / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Plant hybridization --- p.1 / Chapter 1.2 --- Hybridization barriers --- p.2 / Chapter 1.3 --- Technologies to overcome hybridization barriers --- p.2 / Chapter 1.4 --- CENH3 and chromosome elimination --- p.3 / Chapter 1.4.1 --- Chromosome elimination --- p.3 / Chapter 1.4.2 --- Centromere and CENH3 --- p.5 / Chapter 1.4.3 --- Hypothesis of CENH3 and chromosome elimination --- p.9 / Chapter 1.5 --- Experiment design --- p.9 / Chapter 1.5.1 --- Wheat transformation --- p.10 / Chapter 1.5.2 --- Somatic hybridization --- p.13 / Chapter 1.5.3 --- Microprotoplast fusion --- p.15 / Chapter 1.6 --- Applications of somatic hybridization --- p.17 / Chapter 1.6.1 --- Plant breeding --- p.17 / Chapter 1.6.2 --- Minichromosome technology --- p.18 / Chapter Chapter 2 --- Materials and methods --- p.23 / Chapter 2.1 --- Genetic transformation of wheat --- p.23 / Chapter 2.1.1 --- Wheat growth and callus induction --- p.23 / Chapter 2.1.2 --- Constructs for wheat transformation --- p.24 / Chapter 2.1.3 --- Plasmid DNA extraction --- p.26 / Chapter 2.1.4 --- Gold particle bombardment and plants regeneration --- p.26 / Chapter 2.1.5 --- Seed setting rate of transgenic wheat plants --- p.27 / Chapter 2.2 --- Identification of transgenic wheat plants --- p.28 / Chapter 2.2.1 --- PCR screening of transgenic plants --- p.28 / Chapter 2.2.2 --- Fluorescence microscopy and imaging --- p.31 / Chapter 2.2.3 --- FISH of transgenic wheat calli --- p.32 / Chapter 2.3 --- Wheat x maize hybridization --- p.33 / Chapter 2.3.1 --- Transgenic maize plants with miniB chromosomes --- p.33 / Chapter 2.3.2 --- Non-transgenic and transgenic wheat growth and wheat x maize hybridization --- p.34 / Chapter 2.3.3 --- Embryo rescue and cytological analysis of hybrids --- p.34 / Chapter 2.4 --- Wheat x maize somatic hybridization --- p.35 / Chapter 2.4.1 --- Wheat callus culture --- p.35 / Chapter 2.4.2 --- Maize callus culture --- p.36 / Chapter 2.4.3 --- Cell suspension cultures --- p.36 / Chapter 2.4.4 --- Protoplast isolation from wheat and maize --- p.37 / Chapter 2.4.5 --- Microprotoplast isolation --- p.41 / Chapter 2.4.6 --- Protoplast fusion, culture and regeneration --- p.43 / Chapter 2.4.7 --- Cytological analysis of fusion protoplasts --- p.45 / Chapter 2.4.8 --- FISH of fusion protoplasts --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Transgenic wheat with fusion CenH3 gene --- p.47 / Chapter 3.1.1 --- Generation of transgenic wheat plants --- p.47 / Chapter 3.1.2 --- PCR screening of transgenic wheat --- p.49 / Chapter 3.1.3 --- Dwarf mutant in transgenic wheat --- p.51 / Chapter 3.1.4 --- Localization foreign CENH3 by fluorescent protein tag --- p.54 / Chapter 3.1.5 --- Transgene segregation in T₂ generation and callus induction from T₂ transgenic plants --- p.54 / Chapter 3.1.6 --- Detection of transgene by FISH --- p.57 / Chapter 3.1.7 --- Transgenic plants expressing foreign CENH3 protein have lower seed setting --- p.59 / Chapter 3.2 --- Genetic cross of transgenic wheat x maize --- p.61 / Chapter 3.2.1 --- Embryo rescue of zygotic hybrids --- p.61 / Chapter 3.2.2 --- Cytological examination of rescued events --- p.62 / Chapter 3.3 --- Somatic hybridization of transgenic wheat x maize --- p.64 / Chapter 3.3.1 --- Establishment of wheat suspension cell, protoplast and microprotoplast isolation --- p.64 / Chapter 3.3.2 --- Establishment of maize suspension cell, protoplast and microprotoplast isolation --- p.67 / Chapter 3.3.3 --- Somatic hybridization --- p.70 / Chapter 3.3.4 --- Cytological examinations of somatic hybrids --- p.72 / Chapter Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Selectable marker genes for wheat transformation --- p.78 / Chapter 4.2 --- Fragmentation of transformed DNAs --- p.79 / Chapter 4.3 --- Wheat and maize CENH3 --- p.81 / Chapter 4.4 --- Embryo rescue by callus induction --- p.83 / Chapter 4.5 --- Plant microprotoplast isolation --- p.84 / Chapter 4.6 --- Maize protoplast isolation and cell suspension culture --- p.86 / Chapter 4.7 --- Maize B-repeat hybridizes to wheat telomere region --- p.87 / Chapter 4.8 --- Fusion CENH3 could not eliminate abnormal chromosome behaviors in somatic hybrids --- p.88 / Chapter Chapter 5 --- Conclusions --- p.89 / References --- p.90

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