Tesi: "Protoplast fusion"

1

Illing, G. T. "Protoplast fusion and regeneration in Streptomyces clavuligerus". Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378980.

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2

Mandegaran, Zohreh. "Genetic improvement of roses by protoplast fusion". Thesis, University of East London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339583.

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3

Brites, Anny Stella Monteiro. "Seleção de linhagens de Saccharomyces cerevisiae potencializadas pelo fator Killer, H2S- e o carater floculante". Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/11/11141/tde-19052003-144728/.

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Dentre as características desejáveis em leveduras fermentadoras alcóolicas estão a capacidade de floculação, a não produção de H2S e o caráter "killer". Neste trabalho foram selecionadas sete linhagens de Saccharomyces cerevisiae com algumas destas características, que passaram por testes confirmativos e pela cariotipagem eletroforética resultando na escolha de duas linhagens: ATCC 26602 (altamente floculante) e K1 (H2S - e possuidoras do caráter "killer"). Estas linhagens foram utilizadas em um cruzamento via fusão de protoplasto para se obter um produto de fusão estável com as características de interesse tecnológico. Na seleção das linhagens híbridas com base em caracteres naturais foram isolados 1291 híbridos em meio seletivo e entre essas colônias somente 1,5% foram inicialmente consideradas híbriadas. Após três subcultivos em YEPD líquido, estes produtos de fusão não se mostraram estáveis.
Flocculative and "killer" skills and lack in production of H2S are desirable characteristics of the ethanolic fermentative yeasts. Seven selected strains of Saccharomyces cerevisiae with some of these characteristics were evaluated for confirmation of these habilities and their genetic characterization was undertaken by eletrophoretic kariotyping. The strain ATCC 26602 had flocculant hability and the strain K1 was H2S - and "killer". The strains were selected for protoplast fusion aiming to obtain a stable fusion strain with these desirable technologyc characteristics. The selection of the hybrid strains were based on natural characters and have shown 1291 hybrids (frequency of 1,5%) in the medium for the isolation of the fusionants (protoplasts). The protoplast stability were monitored by three continuous growth in the YEPD liquid midium and the stable fusion products were not obtained.

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4

Maren, Nathan Allen. "Symmetric Protoplast Fusion in Interserial Syringa (Oleaceae) Hybridization". Thesis, North Dakota State University, 2016. https://hdl.handle.net/10365/28026.

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Few other woody plants embody the preeminence of temperate woody plants in garden cultivation like the lilacs. In spite of their relationship, the trees lack the diversity of cultivated floral forms observed within the shrub lineages. Typical selection and cross-pollination schemes within the tree lilacs or between trees and shrubs have failed to yield the diversity of colors and fragrances on a tree form. With somatic fusion in Citrus spp. as a guideline for Syringa spp. protoplast isolation and culture, experiments were designed to optimize the conditions through somatic fusion. Protoplast isolation experiments revealed yield increases with increased exposure to cell wall degrading enzymes as well as losses in viability with increased exposure. Electrofusion experiments yielded somatic hybrids, yet further investigation is necessary to optimize the fusion electroporation settings and beyond.

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5

Hansen, Christine S. "Construction of galactose assimilating, carotenoid producing yeasts by protoplast fusion". Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/27935.

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Protoplasts were prepared from two yeast strains P. rhodozyma (ATCC 24202) and K. fragilis (ATCC 8455). Protoplasts prepared from P. rhodozyma were facilitated by prior growth of the cells in a media containing S-(2-aminoethyl)-L-cysteine. Protoplasts from these two yeast genera were fused either by the use of electrofusion or polyethylene glycol treatment. Stable carotenoid producing cell lines were selected by growth at 30°C on yeast nitrogen base plus galactose. Selected single fusants display taxonomic characteristics common to both genera with a cellular morphology and a carotenoid composition similar to that of P. rhodozyma.
Land and Food Systems, Faculty of
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6

Raikar, Sanjeev Vencu. "Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression". Phd thesis, Lincoln University. Bio-Protection and Ecology Division, 2007. http://theses.lincoln.ac.nz/public/adt-NZLIU20080214.105406/.

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Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination A (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination A (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

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7

Raikar, S. V. "Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression". Diss., Lincoln University, 2007. http://hdl.handle.net/10182/301.

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Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

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8

Johnson, Alexander Arthur Theodore. "Protoplast Fusion for the Production of Intermonoploid Somatic Hybrids in Cultivated Potato". Thesis, Virginia Tech, 1998. http://hdl.handle.net/10919/46514.

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Monoploid potato genotypes represent plant material that is free from the "genetic load" of lethal and severely deleterious alleles normally present in the highly heterozygous cultivated potato species. Field evaluations enabled the identification of agronomically superior monoploid potato genotypes from a population of more than 100 anther-derived monoploids. Chemical fusion and electrofusion between pairs selected from 31 superior monoploids resulted in the production of three different groups of intermonoploid somatic hybrids. The hybridity of somatic hybrid plants and calluses was confirmed through PCR-based amplification of simple sequence repeat (SSR) sequences in the potato genome. Polymorphic SSR loci between the monoploid parents of a particular group of somatic hybrids were used to separate true somatic hybrids (heterozygous at the loci) from parental somaclones regenerating from unfused protoplasts (homozygous for one parental band at the loci). One group of somatic hybrids (SH1, SH2 and SH2B) was of particular interest because it resulted from the fusion of a S. phureja monoploid to a high acetylleptinidine-producing monoploid derived from an F1 hybrid between S. chacoense and S. phureja. The leptine acetylleptinidine (ALD) is produced only by some accessions of S. chacoense Bitt. and provides resistance to feeding by the Colorado potato beetle (Leptinotarsa decemlineata Say) when present in sufficient concentrations. The somatic hybrids produced moderate levels of ALD in leaves and stems (roughly 60% that of a high ALD-producing S. chacoense clone). Pollinations of SH1, SH2 and SH2B by several diploid and tetraploid potato clones resulted in three fruit on SH2, one fruit on SH2B and no fruit on SH1. Two resulting progeny populations of SH2 [SH2A = SH2 × S. andigena 8-1 (4x); SH2P = SH2 × S. phureja 66AP11-53 (2x)] expressed higher fertility than the original somatic hybrids and were sexually crossed as both male and female parents to S. tuberosum cv. Atlantic. All of the SH2 progeny populations expressed acetylleptinidines, albeit at lower levels than the SH2 somatic hybrid, providing strong evidence that the genes controlling acetylleptinidine production are dominant. Variation for ALD expression in the SH2 progeny indicated one or a few genes with additive effect controlling the ALD trait. In addition, the choice of male parent in sexual crosses to SH2 affected subsequent ALD expression in progeny populations. The SH2 progeny represent an important first step towards transferring acetylleptinidines to cultivated potato. SH1, SH2 and SH2B appeared to be negatively affected by an unusually high ploidy (hexaploid, 6x). Field-grown plants produced many tubers (mean = 35) of low weight (mean = 10.4 g) and were stunted in appearance. Anther culture of SH2 yielded triploid regenerants (3x). These regenerants may be more phenotypically normal than the original somatic hybrids because of lower ploidy. Segregation of SSR alleles in the triploid anther culture regenerants provided evidence that the hexaploid somatic hybrid SH2 genome is comprised of four homologous genomes of CP2-103 (the high leptine-producing monoploid) and two homologous genomes of 13-14 203 (the S. phureja monoploid).
Master of Science

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9

Hothersall, Joanne. "Metabolite production and molecular characterisation of interspecific Aspergilli". Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285775.

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10

Ravichandran, Vidya. "Application of molecular markers to characterize potato plants derived from anther culture and protoplast fusion". Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-11072008-063210/.

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11

Bennett, Robert Ian. "The development of techniques for the selection of somatic hybrids of Brassica spp. produced by protoplast fusion". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334116.

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12

McCutchan, Jennifer Susan. "Transferring ascochyta blight resistance from Lathyrus sp. into field pea (Pisum sativum L.) via protoplast fusion (somatic hybridisation) /". Connect to thesis, 2001. http://eprints.unimelb.edu.au/archive/00000696.

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13

Lightbourn, Gordon James. "Development of intermonoploid somatic hybrids of potato and their molecular analysis based on polymorphism for retroelement Tst1". Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/28810.

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Inbred lines for hybrid crop production have been a mainstay of plant breeding. Biotechnological approaches to hasten the process are available including anther culture to halve the genome and protoplast fusion to create hybrids between incompatible partners. We applied these techniques to potato to evaluate their potential for breeding highly heterozygous, cross-pollinating species. Four families of monoploids (2n=1x=12), developed from diploid hybrids with diverse genomic constitutions but heavily favoring Solanum phureja, a primitive cultivated potato, were used in electrofusion experiments to create intermonoploid somatic hybrids (SH). The "monoploid sieve" results in the survival of only those gametes free of lethal and deleterious genes but generates sterile sporophytes, necessitating protoplast fusion for SH development. From six intermonoploid electrofusion combinations, 276 plants were regenerated over 6-9 months. Fusion conditions were optimized. Ploidy was determined by flow-cytometry and SH confirmed by microsatellite analysis. Field evaluations over three years revealed that intermonoploid SH were inferior to cultivars. Dihaploids derived by anther culture of a tetraploid intermonoploid SH were reduced in vigor with an increase in homozygosity, while 2x X 2x sexually derived populations had better yield than the SH, suggesting that producing SH introduced or eliminated factors required for productivity. Molecular analysis of the SH was conducted to examine genomic stability through protoplast isolation and plant regeneration. Sequence specific amplified polymorphism (S-SAP) represents a hybrid system incorporating amplified fragment length polymorphism (AFLP) technology in conjunction with the use of a defined genomic sequence, e.g., retrotransposon display (RD) when the defined sequence is anchored into a consensus sequence of a retrotransposon such as the long terminal repeat (LTR) sequence of Tst1. Parental monoploids, SH and various Solanaceae were evaluated by RD. Fluorescently-labeled retrotransposon-based primers were used in the ALFexpress automated fragment analyzer system. Eleven probes from RD were created for Southern blot analysis and used to verify taxonomic relationships between selected Solanaceae. Blots of intermonoploid somatic hybrids confirmed hybridity and occasional loss of genomic fragments. No activation or replication of retrotransposons was detected. Sequencing of inter-retrotransposon amplified polymorphism (IRAP) and S-SAP fragments revealed that all fragments had the expected Tst1 retroelement and/or the AFLP adaptor sequence. BLAST analysis identified 4 of the 17 fragments sequenced as part of the chloroplast genome, a tobacco anther-specific gene, repetitive DNA, and the phytochrome F gene.
Ph. D.

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14

Calixto, Marcia Cristina. "Hibridação somática entre Citrus sinensis e C. grandis". Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/11/11136/tde-29072003-083246/.

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A hibridação somática de citros tem sido extensivamente aplicada, favorecendo o desenvolvimento de híbridos somáticos em programas de melhoramento genético, como fonte de germoplasma ou como variedades copa e porta-enxerto. Neste contexto, este trabalho foi desenvolvido com o objetivo de selecionar plantas de toranja (Citrus grandis L. Osbeck) tolerantes à Phytophthora sp. e utilizá-las como parentais no processo de hibridação somática com outras espécies do gênero Citrus, a fim de produzir híbridos somáticos para o melhoramento de porta-enxertos. Plantas de 20 variedades de toranja tolerantes à Phytophthora spp. foram selecionadas, após serem cultivadas em solo infestado. Experimentos de fusão de protoplastos foram realizados envolvendo laranjas doces, tangerinas e o tangor Murcote, como parentais embriogênicos, e variedades de toranja selecionadas e plantas enxertadas de 12 variedades de toranja, como parentais não-embriogênicos, utilizando-se a técnica de fusão química, via polietilenoglicol (PEG). Microcolônias foram transferidas para meio de cultura MT semi-sólido, suplementado com 500 mg.l -1 de extrato de malte para indução da embriogênese somática. A confirmação da hibridação somática das plantas regeneradas e aclimatizadas foi realizada por meio de análises de morfologia foliar, de citologia, pela contagem do número de cromossomos, e moleculares, por marcadores do tipo RAPD. As metodologias utilizadas para seleção de plantas matrizes, fusão de protoplastos, regeneração de plantas e confirmação da hibridação somática foram adequadas, e permitiram a obtenção de híbridos somáticos de laranja Hamlin com toranja enxertada Indian Red e com seedling selecionado de toranja Singapura, que apresentam potencial para serem incorporados em programas de melhoramento de porta-enxertos.
Citrus somatic hybridization has been extensively applied assisting the development of the somatic hybrids which can be used in improvement programs, indirectly as germoplasm source or directly as scion and rootstock varieties. In this context, this research was developed with the objective of selecting plants of pummelo (C. grandis L. Osb) tolerant to Phytophthora sp. and use these plants as parents in the somatic hybridization process with other species of Citrus. Plants of 20 pummelo varieties, tolerant to Phytophthora sp., were selected after being grown in infested soil. Protoplast fusion experiments were induced by chemical method, with polyethylene glicol (PEG), involving sweet oranges, mandarins and Murcott tangor, as embryogenic parents, selected pummelo varieties and grafted plants of 12 pummelo varieties, as non-embryogenic parents. Microcolonies were transferred to EME semi-solid MT containing 500 mg.l -1 of malt extract for somatic embryogenesis. Somatic hybridization was confirmed by analysis of leaf morphology, citology by chromosome counting and molecular analysis by RAPD markers. The protocols used to select plants to be used as protoplast source, protoplast fusion, plant regeneration and somatic hybridization confirmation were adequate, allowing to produce somatic hybrids of Hamlin sweet orange with Indian Red grafted pummelo and Singapura pummelo selected seedling, which may be used as rootstocks and incorporated in rootstocks improvement programs.

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15

Hennig, Anne [Verfasser], Andrea [Akademischer Betreuer] [Gutachter] Polle e Reiner [Gutachter] Finkeldey. "Drought stress response of tetraploid hybrid aspen (Populus tremula L. x P. tremuloides Michx.) of protoplast fusion experiments) / Anne Hennig. Betreuer: Andrea Polle. Gutachter: Andrea Polle ; Reiner Finkeldey". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1102535656/34.

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16

Souza, Bárbara Lizandra Perini de. "Fusão de protoplastos entre Penicillium echinulatum e Trichoderma harzianum para obtenção de variabilidade visando a produção de celulases". reponame:Repositório Institucional da UCS, 2007. https://repositorio.ucs.br/handle/11338/881.

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O estudo de fungos celulolíticos tem-se mostrado relevante, tendo em vista o interesse econômico do complexo celulases, especialmente na indústria têxtil e, mais recentemente, para propósitos energéticos. No presente trabalho, a fusão de protoplastos foi utilizada para combinar genótipos de mutantes parcialmente desreprimidos para produção de celulases de Penicillium echinulatum (9A02S1B9) e richoderma harzianum (AS5CH3), utilizando a técnica do doador morto, buscando-se obter recombinantes com maior produção de celulases. Nesta estratégia, ambas as linhagens tiveram seu micélio tratado com Glucanex 0,01 g/mL, para quebra da parede celular. Os protoplastos resultantes da linhagem portadora de marca de resistência ao benomil (9A02S1B9) foram inativados por calor (técnica do doador morto) de 60oC antes da etapa de fusão, a qual após foi induzida por PEG4000 e Ca2+, com protoplastos da linhagem sensível ao benomil (AS5CH3). A partir de um produto de fusão, foram selecionados 24 sub-clones, após estratégias de estabilização e seleção para precocidade e eficiência na formação de halo de hidrólise de celulose em placas de Petri. Os produtos de fusão apresentaram morfologia e esporulação semelhantes a um dos parentais, sendo treze semelhantes à Penicillium, nove semelhantes à Trichoderma e dois mostrando formas alteradas. Os produtos de fusão que segregaram para morfologia de T. harzianum apresentaram a característica de resistência ao benomil, sendo capazes de crescer e esporular em meios contendo até 100 μg/mL deste inibidor. A morfologia, o perfil de bandas, obtidos por RAPD, e o padrão de secreção de celulases dos produtos de fusão foram sempre mais semelhantes a um dos parentais. Os clones apresentaram variação quanto ao halo de hidrólise de celulose em placas de Petri e na atividade sobre papel filtro FPAases, -glicosidase ou endoglicanase, quando crescidas em cultivo submerso ou em estado sólido. Desta variabilidade, verificaram-se aumentos significativos para algumas das linhagens em relação aos parentais. A aplicação da metodologia de fusão de protoplastos para obter recombinantes entre P. echinulatum e T. harzianum, empregando a técnica do doador morto, mostrou-se adequada na geração de variabilidade para produção de celulases.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.

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17

Bayley, C. C. R. "An assessment of the consequences of fusion of tobacco and alfalfa protoplasts". Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376160.

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18

Ftouhi, Nouzha. "Étude de la recombinaison génétique par fusion de protoplastes de Bacillus subtilis". Paris 11, 1989. http://www.theses.fr/1989PA112072.

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Abstract (sommario):

La fusion des protoplastes de Bacillus subtilis, décrite pour la première fois par Schaeffer, Cami et Hotchkiss, permet la formation de recombinants génétiques pésentant des combinaisons nouvelles des marqueurs dérivés de deux lignées auxotrophes parentales différentes. Ce type d'échange génétique permet ainsi d'étudier la recombinaison dans la totalité du génome bactérien. Un mélange de protoplastes dérivant de deux souches parentales mutées dans différentes régions du chromosome, à été traité par le polyéthylène glycol et les produits de régénération de la paroi des protoplastes ont été analysés. Les résultats, en absence de pression de sélection ont montré que l'intervalle contenant l'origine de réplication du chromosome, présente la fréquence la plus élevée d'échanges génétiques. Un des points principaux de nos recherches a été de déterminer le rôle du produit du gène rec E dans les échanges observés. Il apparait que des évènements de recombinaison spécifique de l'origine de réplication du chromosome sont indépendants de l'activité RecE. De même, une corrélation entre les fonctions de réplication du chromosome et la recombinaison génétique a été mise en évidence. Dans la dernière partie de ce travail nous avons observé que les changements dans le profil de méthylation de l'ADN stimule la recombinaison homologue par un mécanisme totalement dépendant de l'activité RecE
Fusion of B. Subtilitis, protoplasts, first described by Schaeffer, Cami and Hotchkiss, allows the formation of genetic recombinants showing new combinations of markers derived from two different auxotrophic parent lines. This manner of gnetic exchange permits the study of recombination in the entire genome. A mixture of protoplasts derived from bacterial strains marked genetically by mutation in multiple chromosome regions was treated with polyethylene-glycol and the wall regenerated products were analysed. In the absence of any selective pressure, it was oberved that the genetic interval comprising the origin of chromosome replication show the highest level of genetic exchange. One of the main emphases of the research was to elucidate the role of the rec E gene product in the exchanges observed. It appears that some "origin-specific" recombinational events are dependent of Rec E protein activity. Also the correlation between the replication function of the chromosome and the process of genetic recombination is shown. In the last part of this work a correlation of the DNA methylation pattern changes and the stimulation of homologous recombination was found

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Nagar, Pankaj. "Fusion de protoplastes de Bacillus subtilis : étude de l'apparition de l'inactivation chromosomique et de la structure génétique des diploïdes non complémentants stables". Paris 11, 1985. http://www.theses.fr/1985PA112300.

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L’inactivation chromosomique observée dans les bactéries diploïdes résultent de fusion de protoplastes de deux souches poly auxotrophes de B. Subtilis nécessite la régénération de la paroi Dans ces diploïdes désignés non complementing diploids ou Ncd, il existe des facteurs responsables de cette inactivation. Dans ces diploïdes, l’inactivation chromosomique peut affectés en plus d’un chromosome entier, une séquence du chromosome exprimé
The chromosome inactivation observed in the diploids resulting after protoplast fusion of two polyauxotrophic strains of B. Subtilis, requires the regeneration of the [cellvall]. These diploids called non complementing diploids or ‘Ncd’ carry the factor(s) implied in this inactivation can affect a sequence of expressed chromosome in addition to one [entise] chromosome

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20

Dornelas, Marcelo Carnier. "Cultura e fusão de protoplastos de Passiflora spp". Universidade de São Paulo, 1995. http://teses.usp.br/teses/disponiveis/11/11137/tde-20181127-160654/.

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Programas de melhoramento genético de maracujá (Passiflora edulis var. flavicarpa Deg.) têm sido conduzidos no Brasil visando a seleção de variedades mais produtivas. Híbridos interespecíficos entre a espécie cultivada e espécies selvagens relacionadas têm sido obtidos, usando técnicas de hibridação sexual, na tentativa de transferir genes de resistência à doenças para o híbrido. Entretanto estes híbridos interespecíficos possuem problemas de fertilidade. Isto dificulta seu aproveitamento em programas de melhoramento e justifica o uso da técnica de hibridação somática. Esta dissertação descreve o desenvolvimento de protocolos para o isolamento e a cultura de protoplastos de espécies de Passiflora. O procedimento resulta na regeneração de plantas inteiras, bem como na obtenção de hídricos somáticos resultantes da fusão de protoplastos isolados da espécie comercial e de espécies selvagens de Passiflora. Protoplastos foram isolados de explantes foliares de P. alata Ait., P. amethystina Mikan, P. cincinnata,/i> Mast., P. coccínea Aubl., P. edulis var. flavicarpa Deg. E P. giberti N.E. Brown, mediante tratamento enzimático. Os protoplastos mostraram-se capazes de regenerar a parede celular, sofrer divisões e formar colônias. Em condições apropriadas, as colônias desenvolveram calos e regeneraram brotos. Plantas inteiras puderam ser obtidas de todas as espécies estudadas, com exceção de P. coccínea. Protoplastos isolados de tecido foliar de P. edulis var. flavicarpa e de suspensões celulares das espécies selvagens, foram fundidos com o uso de uma solução contendo 30% (p/v) de PEG. O cultivo dos produtos de fusão resultou em 3 a 5% de calos híbridos. Estes foram selecionados pelo padrão eletroforético de bandas de proteínas solúveis e de isoenzimas. Os híbridos somáticos de P. edulis var. flavicarpa (+) P. alata, P. edulis var. flavicarpa (+) P. amethystina, P. edulis var. flavicarpa (+) P. cincinnata e P. edulis var. flavicarpa (+) P. giberti, foram regenerados desenvolveram-se em plantas inteiras, que apresentaram 4n=36 cromossomos.
not available

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Carvalho, Dayse Cristina de. "Fusão de protoplastos visando a reconstrução da laranja azeda". Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/11/11136/tde-12012007-160405/.

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O objetivo deste trabalho foi aplicar a técnica de fusão química de protoplastos para desenvolver híbridos somáticos interespecíficos entre tangerinas (Citrus reticulata) e toranjas (Citrus grandis), visando a obtenção de porta-enxertos semelhantes à laranja azeda Citrus aurantium). Como fonte de protoplastos foram utilizadas suspensões celulares embriogênicas de tangelo \'Page\' (C. reticulata x C. paradisi) e tangor \'Murcote\' (C. reticulata x C. sinensis) e folhas jovens de seedlings de toranjas \'Lau Tau\' e \'Ogami\' (Citrus grandis). Após a fusão os protoplastos foram cultivados sob ausência de luz, até a formação de microcolônias, que foram então cultivadas em meio de cultura EME em dupla-fase, suplementado com 13,33 g/L de maltose para a indução da embriogênese. Os embriões globulares formados foram transferidos para meio EME com 25 g/L de sacarose e quando em estádio cotiledonar foram transferidos para meio de cultura suplementado com 1,5 g/L de extrato de malte. As brotações obtidas foram enxertadas in vitro sobre laranja \'Hamlin\' e \'Valência\'. As plantas obtidas foram levadas para casa de vegetação e cultivadas em substrato comercial. As 17 plantas regeneradas de tangelo \'Page\' + toranja \'Lau Tau\' apresentaram conformação fenotípica adversa aos genitores, com folhas de tamanho reduzido, ápice arredondado, coloração verde escura, mesófilo enrugado e ausência de pecíolo alado. A análise de citometria de fluxo confirmou o caráter diplóide das plantas regeneradas. Marcadores moleculares RAPD apresentaram padrão de bandas similar entre a planta regenerada e o genitor tangelo \'Page\'. O protocolo utilizado para isolamento, fusão e cultura de protoplastos, bem como para a regeneração e aclimatização de plantas permitiram a obtenção 17 plantas da combinação entre tangelo \'Page\' e toranja \'Lau Tau\' com conformação fenotípica diferente dos genitores, duas plantas da combinação entre tangor \'Murcote\' e toranja \'Ogami\' e uma planta da combinação entre tangor \'Murcote\' e toranja \'Lau Tau\'.
The aim of this work was to apply the technique of chemical fusion of protoplasts, in order to develop interspecific somatic hybrids between mandarins (Citrus reticulata) and pummelos (Citrus grandis), in order to produce similar to sour orange (Citrus aurantium). The sources for protoplasts were embryogenic suspension cultures of \'Page\' tangelo (C. reticulata x C. paradisi) and \'Murcott\' tangor (C. reticulata x C. sinensis) and young leaves from seedlings of \'Lau Tau\' and \'Ogami\' pummelos (Citrus grandis). After the fusion, the protoplasts were cultivated in the absence of light, until the formation of microcolonies, and were then cultivated in double-phase EME medium, supplemented with 13.33 g/L of maltose for embryogenesis induction. The globular embryos thus formed were transferred to EME medium with 25 g/L of sucrose and, when in cotyledonal stage, were transferred to a culture medium supplemented with 1.5 g/L of malt extract. The shoots obtained were grafted in vitro onto \'Hamlin\' and \'Valencia\' sweet oranges. The regenerated plants were cultivated in a greenhouse, over commercial substrate. In this process, 17 plants were obtained. These plants presented phenotypic conformation different from the genitors, with leaves with reduced size, round apex, dark green coloration, rough leaf blade and absence of developed petiole. The analysis by flow cytometry confirmed the diploid character of the regenerated plants. RAPD molecular markers presented a similar band pattern between the regenerated specimen and the genitor \'Page\' tangelo. The protocol used for isolation, hybridization and cultivation of protoplasts, as well as for the regeneration and acclimatization of the plants allowed the obtainment of 17 plants from the combination of \'Page\' tangelo + \'Lau Tau\' pummelo, with phenotypic conformation different from the genitors, two plants from the combination of \'Murcott\' tangor + \'Ogami\' pummelo and one plant from the combination of Murcote\' tangor + \'Lau Tau\' pummelo.

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AIT, BARHOUCH JALILA. "Developpement des protoplastes et hybridation somatique chez les ectocarpales (fucophycees). Caracterisation des produits de fusion". Paris 6, 1996. http://www.theses.fr/1996PA066451.

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La mise en oeuvre de l'hybridation somatique chez les fucophycees necessite une bonne maitrise des mecanismes d'isolement, de regeneration des protoplastes et de selection des produits de fusion. Deux ectocarpales filamenteuses, pylaiella littoralis et ectocarpus siliculosus, ont ete choisies pour realiser ce travail grace aux avantages qu'ellespresentent dans des etudes fondamentales et a finalite biotechnologique : structure du thalle simple, cultures axeniques, taux eleve d'isolement et de regeneration des protoplastes. L'optimisation des conditions d'isolement a permis d'obtenir de facon repetitive une grande quantite de protoplastes viables aptes au developpement chez les deux especes. Les modalites de regeneration suivies par les protoplastes de ces deux souches sont identiques. Les protoplastes se divisent et regenerent selon trois modeles differents qui peuvent etre mis en relation avec leur cellule d'origine et leur etat morphogenetique au sein du thalle. Deux techniques, electrique et chimique (peg), ont ete utilisees pour fusionner les protoplastes de p. Littoralis. La chelation par le citrate de sodium des ions calcium presents dans toutes les solutions de traitement des protoplastes est necessaire pour induire la fusion. Seule la methode chimique a permis d'obtenir des produits de fusion viables. Ces derniers sont selectionnes apres regeneration sur des caracteres phenotypiques et analyses par cytometrie en flux. Les histogrammes obtenus apres analyse en cytometrie en flux des niveaux de plodie des produits de fusion revelent des pics correspondant a la population tetraplode majoritaire temoignant que la fusion est effective. Differentes approches ont ete abordees pour obtenir des souches possedant des marqueurs biochimiques ou moleculaires permettant, dans le cadre d'un programme de fusion, de realiser un tri precoce des hybrides putatifs intergeneriques entre p. Littoralis et e. Siliculosus. C'est dans cette optique que nous avons mis en oeuvre, chez les protoplastes de p. Littoralis, la transformation par la strategie biolistique et la mutagenese par irradiation au uv. La technique rapd, developpee chez ces deux ectocarpales, s'est revelee efficace pour discriminer ces especes en fournissant des marqueurs moleculaires detectant un polymorphisme d'adn interspecifique.

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Cordeiro, Antônio Teixeira. "Embriogênese somática indireta e fusão interespecífica de protoplastos em Coffea". Universidade Federal de Viçosa, 1998. http://www.locus.ufv.br/handle/123456789/9981.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Esta pesquisa foi conduzida com o objetivo de verificar a exeqüibilidade da fusão protoplástica interespecífica como ferramenta no melhoramento parassexual em Coffea. Para tanto, pretendeu-se a introgressão das resistências aos nematóides Meloidogyne incognita e M. exigua, do cultivar canéfora Apoatã, nos genótipos arábicas diplóide DH 3 e tetraplóides Catimor e Catuaí Amarelo. Com vistas à obtenção dos protoplastos, foi dado início a suspensões celulares embriogênicas a partir de calos friáveis embriogênicos induzidos de explantes foliares. Posteriormente foram estudadas, nas suspensões celulares Apoatã e DH 3 , as cinéticas do crescimento e do rendimento protoplástico em função do tempo pós-subcultura, bem como as condições de digestão enzimática da parede celular de seus agregados celulares. Foram realizados, ainda, estudos para a seleção dos heterofusionados. Embora todos os genótipos testados tenham reagido favoravelmente à formação de tecido embriogênico friável, apenas os arábicas DH 3 e Catuaí Vermelho requereram a ação conjunta de auxina e citocinina. Os demais Apoatã, Catimor e Catuaí Amarelo reagiram mais favoravelmente quando o regulador de crescimento foi apenas a citocinina BAP. As maiores freqüências de explantes com calos friáveis verificadas para os genótipos canéfora Apoatã e arábicas DH 3 , Catimor, Catuaí Amarelo e Catuaí Vermelho foram, respectivamente, 80, 60, 40, 40 e 20%. A diferenciação embriogênica dos agregados celulares, em cultura líquida, rendeu cerca de 241.000, 72.870 e 121.760 embriões g -1 MF de agregados celulares Apoatã, Catimor e DH 3 , respectivamente. As suspensões celulares Apoatã e DH 3 revelaram, imediatamente após a subcultura, uma fase exponencial de crescimento celular até um ponto de inflexão, a partir do qual as taxas de crescimento reduziram progressivamente para atingir um peso de matéria fresca dos agregados celulares assintótico. Coincidentemente, os menores rendimentos protoplásticos foram observados para tempos pós-subcultura superiores àquele do ponto de inflexão. A pré-plasmólise, a seleção dos agregados celulares de diâmetro inferior a 1 mm e a adição de cisteína 0,83 mM, durante a digestão enzimática, não interferiram no rendimento protoplástico das suspensões celulares Apoatã e DH 3 , ao contrário das concentrações das enzimas pectinases e celulase. De maneira geral, o rendimento protoplástico Apoatã, sempre inferior àquele DH 3 , foi maior quando as duas pectinases, pectoliase e macerozima, estiveram associadas à celulase, as três nas maiores concentrações testadas. Os 12 meios de cultivo de protoplastos testados não permitiram distinguir os protoplastos parentais Apoatã e DH 3 em nível de crescimento dos microcalos. Alternativamente, os inibidores metabólicos iodoacetamida e rodamina, nas respectivas concentrações de 2 e de 0,9 mM, mostraram-se eficazes em inibir a regeneração dos protoplastos parentais em microcalos. Seu aproveitamento permitiu a realização de 23 ensaios de eletrofusão interespecífica, cujos produtos, aparentemente justificados pela complementação metabólica, encontram-se em desenvolvimento com vistas à diferenciação embriogênica e regeneração de plantas. Esta última permitirá análises para verificação do potencial da fusão protoplástica no melhoramento parassexual em Coffea.
The aim of the present work was to evaluate the feasibility of interspecific protoplast fusion as a tool in the parassexual improvement in Coffea. Introgression of nematode resistance from Coffea canephora cv. Apoatã was attempted in diploid (DH 3 ) and tetraploid (Catimor and Catuaí Amarelo) C. arabica genotypes by protoplast fusion. Protoplasts were isolated from embryogenic cell suspensions initiated from leaf explants-derived friable embryogenic calli. For Apoatã and DH 3 cell suspensions the kinetics of growth and protoplast yield was evaluated as affected by the intervals of subcultures and the conditions of enzymatic digestion of the cell walls of their small cell aggregates. Investigations aiming at selection of protoplast heterofused-derived cell aggregates were also accomplished. All genotypes studied reacted favorably to the formation of friable embryogenic calli. C. arabica genotypes, DH 3 and Catuaí Vermelho, however, required the use of both auxin and cytokinin. Conversely, Apoatã, Catimor and Catuaí Amarelo presented similar morphogenetic responses when induction media were supplemented with BAP only. Frequencies of friable embryogenic calli of 80, 60, 40, 40 and 20% were obtained for Apoatã, DH 3 , Catimor, Catuaí Amarelo and Catuaí Vermelho, respectively. Embryogenic differentiation from cell aggregates, in liquid culture, produced about 241.000, 72.870 and 121.760 embryos g -1 FW for Apoatã, Catimor and DH 3 , respectively. Immediately to the subculture, Apoatã and DH 3 cell suspensions revealed a typical sigmoidal growth curve. Coincidentally, the lowest protoplast yields were observed for post-subculture intervals above the inflection point. The pre-plasmolysis, the size of the aggregates (smaller than 1 mm diameter) and the addition of cysteine 0.83 mM, during the enzymatic digestion, did not affect protoplast yield of Apoatã and DH 3 cell suspensions, unlike the concentrations of the pectinases and cellulase enzymes. In general, protoplast yields of Apoatã were always inferior to that DH 3 . High yields were achieved when the two pectinases (pectolyase and macerozyme) were combined to cellulase, at the highest tested concentrations. The twelve protoplast culture media tested did not allow any distinction between parental protoplasts Apoatã and DH 3 , at microcalli growth stages. Alternatively, the metabolic inhibitors iodoacetamide and rhodamine, at the concentrations 2 and 0.9 mM, respectively, were effective in inhibiting microcalli formation from cultured parental protoplasts. Its use allowed the accomplishment of 23 independent interspecific electrofusion assessments whose products led to the development of embryogenic differentiation. Further work will be carried out in order to characterize the putative heterofused regenerants and to examine the potential of somatic hybridization as a tool for parassexual improvement in Coffea.
Tese importada do Alexandria

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LEJARD, FREDERIC. "Modifications phenotypiques obtenues par conjugaison et fusion de protoplastes a partir d'une souche industrielle de streptococcus lactis". Caen, 1985. http://www.theses.fr/1985CAEN2027.

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Ben, Khelifa Khadija. "Recherche des mécanismes d'inactivation chromosomique chez les clones diploides non-complémentants produits par fusion de protoplastes de Bacillus subtilis". Paris 11, 1985. http://www.theses.fr/1985PA112048.

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Certaines bactéries diploïdes issues de la fusion de protoplastes de cellules de B. Subtilis, présentent la suppression phénotypique d'un des deux génômes parentaux et par conséquent une absence de complémentation fonctionnelle. On qualifie ces bactéries de Ncd (non-complementing diploids). Dans le but de rechercher si l'inactivation chromosomique observée chez les bactéries Ncd est corrélée avec une organisation «anormale» du nucléoide et/ou une modification de la structure primaire de l'ADN «éteint», trois approches ont été entreprises. La caractérisation des nucléoides extraits de souches Ncd, portant le prophage 105 sur le chromosome inactif, a montré qu'il n'est pas accessible de purifier de façon homogène, les deux nucléoides parentaux, par sédimentation en gradient de saccharose. La visualisation en microscopie électronique des bactéries Ncd indique qu'il n'y a pas de compartimentation pour les deux types de chromosome dans la cellule. Cependant, la configuration de la membrane cytoplasmique des bactéries Ncd, apparaît différente de celle des cellules haploïdes. L'étude de l'activité transformante d'un génôme inactif a montré que, lorsque le lysat brut d'une colonie Ncd est utilisé comme ADN donneur, les allèles sauvages non-exprimés sont incapables de transformer efficacement les bactéries compétentes. Cependant, cette activité peut être restaurée, après traitement à la protéinase K du lysat ou après purification de l'ADN. Il semblerait qu'une ou plusieurs protéines, attachées au nucléoide non exprimé in-vivo, masque la capacité transformante des marqueurs qui lui sont associés. L'existence de séquences nucléotidiques dans l'ADN inactif pouvant servir de signal pour l'association préférentielle de telles protéines, n'a pas été confirmée. Tout au moins pour trois séquences nucléotidiques examinées et en utilisant comme marqueur spécifique du chromosome "éteint" la séquence du phage 105. L'ensemble de ces résultats semble indiquer que l'inactivation d'un chromosome bactérien entier, n'est pas due à une modification de la structure primaire de l'ADN mais résulte d'une conformation «particulière» du nucléoide.

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Amar, Mohamed. "Diploïdes non-complémentants stables produits par fusion de protoplastes de Bacillus subtilis : activité biologique du chromosome non exprimé". Paris 11, 1985. http://www.theses.fr/1985PA112180.

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Dans ce travail, après fusion de protoplastes (induite par le polyéthylène-glycol), de deux souches polyauxotrophes de Bacillus subtilis, nous avons décrit pour la première fois une nouvelle classe de bactéries : les diploïdes non-complémentants stables (Ncd st) présentant une inactivation chromosomique irréversible. Par la suite, nous avons analysé les propriétés biologiques du chromosome non exprimé extrait de son contexte cellulaire. Nous avons constaté que la stabilisation entraîne la perte de l’activité transformante de l’ADN du chromosome en question. Cette perte n’est pas due à l’incapacité de pénétration de l’ADN inactif dans les cellules réceptrices, lors de la transformation génétique, mais à son accumulation probablement sous forme de complexe ADN-membrane cellulaire, ce qui pourrait empêcher son intégration dans le chromosome de la réceptrice
In this present work, we have described for the first time, a novel class of bacterial diploids: stable non-complementing diploids (Ncd st) resulting from protoplast fusion of two polyauxotrophic strains of Bacillus subtilis. These diploids carry and irreversibly inactivated chromosome. We have analyzed the biological properties of this chromosome after its extraction. We have observed that the stabilization is accompanied by the loss of transforming activity of the DNA under study. This loss is not due to the lack of penetration of the inactive DNA in the recipient cells, but is because of its accumulation, probably under a form of DNA-membrane complex, which in turn, would hinder its integration in the chromosome of the recipient cells

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Viaud, Muriel. "Amélioration génétique des deutéromycètes entomopathogènes du genre Beauveria par fusion de protoplastes : approche moléculaire de la recombinaison mitotique". Lyon 1, 1997. http://www.theses.fr/1997LYO10015.

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Les champignons du genre beauveria presentent une grande biodiversite. Ainsi, la souche b. Bassiana bb28 est pathogene d'ostrinia nubilalis, tandis que la souche b. Sulfurescens bs2 n'est pas pathogene mais secrete une entomotoxine puissante. L'amelioration genetique de beauveria a ete envisagee par la fusion de protoplastes. Des outils moleculaires ont ete developpes pour etudier la variabilite chez les souches parentales et pour suivre les genomes parentaux lors des fusions. Les techniques d'amplifications genique et de polymorphisme de longueueur des fragments de restriction ont permis d'obtenir des marqueurs incluant sept genes conserves dont pr1 codant pour une proteinase impliquee dans la penetration de la cuticule. L'etude des regions telomeriques des chromosomes de beauveria, avec une sequence telomerique clonee chez botrytis cinerea, a montre leur interet pour des etudes de structure des populations et de flux de genes. La mise au point de l'electrophorese en champ pulse chez beauveria a permis de mettre en evidence la flexibilite importante du genome. Des fusions de protoplastes entre bb28 et bs2 ont ete realisees dans le but d'obtenir des hybrides aux caracteristiques pathogenes et toxinogenes combinees. Parmi les hybrides interspecifiques stables obtenus, certains se sont averes hypervirulents validant l'interet de la fusion pour l'amelioration des champignons entomopathogenes. La caracterisation moleculaire des hybrides temoigne : (i) de l'intervention de la parasexualite, (ii) d'une ploidie proche de la diploidie et (iii) de la variabilite des structures moleculaires. Les caryotypes des hybrides ont indique la possibilite de rearrangements chromosomiques et l'existence de crossing-overs entre les telomeres de bb28 et bs2. Neanmoins, la recombinaison inter-chromosomique ne semble pas generale a l'ensemble du genome probablement du fait des differences genomiques entre b. Bassiana et b. Sulfurescens.

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Simo, Santalla Pablo. "Contribution au développement de techniques pour l'hybridation somatique chez la pomme de terre (Solanum tuberosum) : culture et fusion de protoplastes de clones dihaploïdes, diploïdes hybrides interspécifiques et de l'espèce diploïde S. brevidens". Paris 11, 1988. http://www.theses.fr/1988PA112393.

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Des aspects différents des méthodes d'obtention d'hybrides somatiques de pomme de terre ont été étudiés et développés. Des colonies ont été obtenues à partir de protoplastes de 12 clones diploïdes sur les 13 étudiés. 10 de ces clones ont régénéré des plantes. L'influence de différents facteurs sur la formation de colonies a été mise en évidence: l'osmolarité du milieu pendant l'isolement et la culture; l'emploi de sels pendant l'isolement; la densité de culture initiale; l'emploi d'antibiotiques (en particulier ampicilline); et la composition du milieu de culture. A chaque génotype correspond un type de comportement particulier par rapport à la composition en substances de croissance et à l'utilisation d'acides organiques dans le milieu de culture. Ces comportements sont dépendants de la concentration des macroéléments dans le milieu. Les taux de régénération varient selon le génotype et le milieu choisi. Une variation morphologique relativement importante a été décrite chez des plantes issues de protoplastes de 2 clones différents. Les réponses des clones étudiés aux conditions d'isolement et de culture de protoplastes, ainsi qu'aux milieux de régénération peuvent être utilisées pour la détermination de protocoles de sélection d'hybrides somatiques par complémentation physiologique. Un grand nombre de plantes ont été régénérées à partir d'expériences de fusion par des méthodes chimiques. Une plante présentant des isoestérases caractéristiques des clones parentaux (hybride somatique probable) a été sélectionnée.

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29

Lopez, Frédéric. "Utilisation d'un plasmide pour l'étude de l'expression du génome diploide des NCD stables issus de la fusion de protoplastes de Bacillus subtilis". Paris 11, 1985. http://www.theses.fr/1985PA112263.

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L’utilisation du plasmide pHV 438 a permis de montrer, dans ce travail que : - la partie d’ADN hétérologue pHV 32, au chromosome de Bacillus subtilis, insérée dans la région ThyB-X près du terminus de la réplication, semble ségréger au cours de la stabilisation des Ncd. Cette ségrégation n’est observée que lorsque cette séquence est sur le chromosome inactif. – la modification de l’ADN du chromosome non exprimé des Ncd stables qui a été corrélée avec le maintien de l’inactivation génique, est dépendante de l’hôte. En effet, la transformation dans E. Coli permet de restaurer le pouvoir transformant d’une séquence inactive alors qu’il n’en est pas de même chez B. Subtilis. – l’intégration dans le chromosome non exprimé d’un diploïde stable, par le processus de la transformation génétique, semble se produire avec une fréquence inférieure à celle de l’intégration dans le chromosome exprimé.

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Hassanpour-Estahbanati, Abolghassam. "Régénération de plantes à partir de protoplastes et après hybridation somatique dans le genre lycopersicon". Paris 11, 1986. http://www.theses.fr/1986PA112097.

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Cette étude présente la mise au point d’une technique de régénération et de l’hybridation somatique dans le genre Lycopersicon, afin d’introduire une information génétique de résistance à la salinité et/ou à la sécheresse chez la tomate cultivée : L. Esculentum. La régénération de plantes à partir de protoplastes de L. Pennellii, L. Chilense et L. Esculentum a été obtenu. Les plantes issues d’une expérience d’hybridation somatique interspécifique entre L. Escelentum et L. Pennellii sont analysées. L'étude ’cytologique et morphologique montre que : « ces plantes sont issues de fusion de deux protoplastes et elles présentent des caractères morphologiques des deux parents. » Enfin, une stratégie d’utilisation de ces techniques dans le genre Lycopersion, tant sur le plan fondamental que sur le plan de l’amélioration des plantes, est discutée
This thesis sought to identify a procedure for regeneration and somatic hybridization in the genus Lycopersicon, in order to introduce salt and/or drought resistance from wild species in cultivated tomatoes. The effect of explant, genotype, and physiological stage of protoplasts source plant and culture technique were studied. Plant regeneration was achieved from protoplasts of L. Pennellii, L. Chilense and L. Esculentum. The regenerated plants after an interspecific somatic fusion between L. Esculentum and L. Pennellii were analyzed. Cytological and morphological studies demonstrate that:"These plants are issued from fusion of two protoplasts and they have a morphological intermediate between two parents. " Finally, the use of these techniques in the genus Lycopersicon, for fundamental questions or in plant breeding is discussed

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D'HONT, ANGELIQUE. "Analyse des genomes nucleaire, chloroplastique et mitochondrial de plantes issues de fusion de protoplastes de medicago; etude de la recombinaison de l'adn mitochondrial". Paris 11, 1990. http://www.theses.fr/1990PA112001.

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L'identification des genomes nucleaires, chloroplastiques et mitochondriaux de plantes issues de fusion de protoplastes entre differentes especes de medicago a ete realisee par analyse du polymorphisme des fragments de restriction (rflp). Les trois plantes h1, h2 et h3, issues d'une experience de fusion entre medicago falcata (p1) et medicato sativa (p2) possedent un genome nucleaire hybride constitue de l'addition des deux genomes parentaux; leur genome chloroplastique est identique a celui du parent p1 et leur genome mitochondrial presente des rearrangements par rapport aux genomes parentaux. Dans une deuxieme partie, nous avons etudie ces remaniements de adn mitochondrial (mt). Nous avons donc construit une banque de l'adnmt de p2 et de h3 dans le site sall du cosmide phc79. Par hybridation moleculaire et cartographie, nous avons mis en evidence, une recombinaison intergenomique entre les deux adnmt parentaux. Cette recombinaison a eu lieu au niveau d'une sequence homologue, localisee chez les deux parents en amont d'une copie du gene alpha-atpase. Chacun des genomes mitochondrial parental contient 2 copies de ce gene, l'hybride h3 en contient trois. Des hybridations northern ont montre que la sequence homologue, ayant donne lieu a la recombinaison, est transcrite et vraisemblablement co-transcrite avec le gene alpha-atpase chez p1, p2 et h3. La transcription du gene alpha-atpase ne semble pas modifiee par la recombinaison. Par contre le profil de transcription de la sequence homologue est modifie chez l'hybride h3

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32

WATIN-DE, PONTFARCY CHRISTINE. "Embryogenese somatique et culture de protoplastes pour la carotte, le persil et le celeri. Experiences preliminaires de fusion somatique en presence d'inhibiteurs metaboliques". Paris 11, 1990. http://www.theses.fr/1990PA112067.

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Le transfert d'une sterilite male de nature cytoplasmique, deja connue, de la carotte au persil ou au celeri pourrait permettre l'obtention d'hybrides f#1 jusqu'alors inexistants pour ces 2 especes. La voie de la fusion de protoplastes a ete retenue, puisque ces especes sont sexuellement incompatibles. Prealablement a ces experimentations, le comportement in vitro du persil et du celeri a ete etudie. Les modalites de la germination in vitro, de la callogenese et de la culture de cellules ont ete precisees pour chacune des especes receptrices. La regeneration de plantes par embryogenese somatique a partir de cals d'explants primaires a ete observee. Cette capacite est etroitement dependante du type d'explant et de la variete d'origine. Apres vernalisation, ces plantes ont fleuri et produit des akenes viables. Pour la premiere fois, la methodologie d'isolement des protoplastes a partir de cal (persil) ou de suspension cellulaire (celeri) et les conditions de leur induction mitotique ont ete definies. Dans le cas du celeri, la regeneration par embryogenese somatique a partir de protoclones a permis l'obtention de plantes dont le comportement au champ a ete etudie; une forte variabilite a ete mise en evidence. La fusion somatique entre protoplastes de carotte et protoplastes de celeri a ete realisee. Afin de disposer d'un crible de selection precoce permettant la reconnaissance des hybrides somatiques au niveau cellulaire, des inhibiteurs metaboliques de specificite differente (rhodamine 6-g et l'acide iodoacetique) ont ete utilises. Leur influence sur la survie, la division et la regeneration de plantes a ete observee. L'absence de repetabilite de ce type d'experiences rend difficile la mise au point d'une methodologie de selection des produits de fusion basee sur l'emploi de ces molecules

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33

Perronnet, Christine. "Recherche de nouvelles méthodes d'obtention de souches productrices de métabolites secondaires par hybridation somatique : fusion de protoplastes de catharanthus roseus ; mise au point d'une technique d'électrofusion ; enrichissement et essais de culture des produits hybrides". Tours, 1989. http://www.theses.fr/1989TOUR3801.

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34

Monteiro, Claudia Barros. "Avaliação da instabilidade mitótica de Aspergillus nidulans através de técnicas genéticas clássicas e fusão de protoplastos". Universidade de São Paulo, 1990. http://teses.usp.br/teses/disponiveis/11/11137/tde-20181127-155509/.

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A instabilidade genética observada no fungo filamentoso Aspergillus nidulans, é semelhante em muitos aspectos aos estudos em milho de McCLINTOCK (1951), com os elementos genéticos móveis. Muitos trabalhos vêm sendo realizados desde 1960 no sentido de caracterizar através da genética clássica, e mais recentemente com as técnicas modernas, o fenômeno de transposição. A instabilidade deste fungo é observada na forma de setores produzidos por uma linhagem que apresenta uma duplicação de um segmento do grupo de ligação I translocado para o grupo de ligação II. Tais setores originados por deleções, novas duplicações, outros rearranjos e por transposição podem ser classificados em melhorados, deteriorados e heterocarióticos. Este trabalho pretendeu ser mais uma contribuição aos estudos básicos dessa instabilidade. Inicialmente através do isolamento de setores deteriorados e sua análise genética via metodologia clássica, já que tais setores podem ser originados por transposição. Foram analisados 7 variantes deteriorados através do ciclo sexual e parassexual, sendo que os determinantes de deterioração foram localizados nos grupos de ligação III IV. Utilizando-se de técnicas modernas através da obtenção, fusão e regeneração de protoplastos, a análise genética dos variantes foi feita em diplóides obtidos por fusão. Nenhuma alteração foi detectada quanto a loca1ização dos determinantes genéticos de deterioração na análise mitótica dos 7 variantes. E finalmente foi verificado se ocorre transposição pela fusão citoplasmática, através da fusão dos protoplastos. Foi encontrado 1 variante hap1óide com fenótipo deteriorado e marcadores genéticos da linhagem MSE, pela fusão de protoplastos desta linhagem com o variante V94, em menos que 600 colônias analisadas. Para se alcançar esses três objetivos, outros pequenos ensaios complementares foram feitos, como a segregação meiótica dos cruzamentos entre variantes deteriorados e a linhagem MSE, em meio com estabilizador osmótico, para diminuir a desvantagem seletiva dos deteriorados em relação as colônias normais; porcentagem de regeneração e fusão de protoplastos, um estudo comparativo das linhagens A, MSE e variantes deteriorados; verificação da toxicidade do PEG aos variantes deteriorados; efeito do fungicida banlate sobre a instabilidade dos diplóides obtidos pela metodologia clássica e fusão de protoplastos; e padrão eletroforético das enzimas do sistema esterase das linhagens A, MSE e variantes deteriorados.
The genetic instability found in the filamentous fungus Aspergillus nidulans is, in many aspects, similar to the studies of moving genetic elements in maize, done by McCLINTOCK (1951). Since 1960, many studies have been carried out with the aim of understanding the phenomena of transposition, initially by using classical genetic techniques and more recently by using modern techniques. The instabili1y of this fungus is observable by the production of sectors by strains which possess a duplicated segment of linkage group I translocated to linkage group II. Such sectors, originated either by deletions, new duplications, transposition or by other rearrangements, may be classified as improved, deteriorated or heterokaryotic. This work is a contribution to the basic studies of this instability, and is divided into three main parts. Initially, deteriorated sectors were isolated and genetica11y analyzed by classica1 methods, since such sectors may be originated by transposition. Seven deteriorated variants were analyzed by their sexual and parasexual cycles. The determinants of deterioration were found to be localized in linkage group III and IV. Modern techniques, namely the obtainment, fusion and regeneration of protoplasts, were used and genetical analysis of variants were carried out on diploides obtained by fusion. No alteration of the 1ocalization of the genetic determinants of deterioration was detected by mitotic analysis of the seven variants. Finally, protoplast fusion was used to verify if transposition occurs from citoplasmic fusion. A haploid variant, with a deteriorated phenotype and genetic markers of the MSE strain, was found from the protoplastic fusion of this strain with the variant V94, within less than 600 analyzed colonies. To achieve the above objectives, other small complementary assays were done, such as the meiotic segregation of the crosses between deteriorated variants and the MSE strain, in a medium containing osmotic stabilizer, with the aim of decreasing the selective desadvantage of the deteriorates; a comparative study of strains A and MSE and deteriorated variants as to percentage of regeneration and fusion of protoplasts; examination of PEG toxicity to deteriorated variants; effect of benlate fungicide upon the instability of the diploids obtained by classical methodology and by protoplast fusion; and the eletrophoretic pattern of the enzymes of the esterase system of strains A and MSE and deteriorated variants.

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Cabasson, Cécile. "Régénération de la mandarine commune (Citrus deliciosa Ten. ) par embryogénèse somatique en milieu liquide : Fusions somatiques et essais de transformation génétique". Montpellier 2, 1993. http://www.theses.fr/1993MON20257.

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Un procede de culture cellulaire en milieu liquide a ete developpe pour une espece modele, la mandarine commune (citrus deliciosa ten. ). La recherche des facteurs permettant d'orienter a volonte les cultures cellulaires de la multiplication indifferenciee vers l'embryogenese somatique, a montre que la diminution de la densite cellulaire au moment de l'inoculation est 60 fois plus efficace que le remplacement du saccharose par le galactose. La difference de comportement des cellules, placees sur galactose ou sur saccharose, se determine tres tot comme en temoignent les differences de consommation de 4 principaux macroelements (calcium, phosphore, potassium, magnesium). La modification des conditions de l'environnement hydrique, avec l'utilisation de systeme a immersion temporaire, favorise la morphogenese des embryons et leur aptitude a la germination. Disposant d'un systeme de culture cellulaire efficace, les manipulations genetiques ont pu etre envisagees. Deux techniques complementaires ont ete abordees: fusion de protoplastes et transfert de genes dans des protoplastes, tous deux induits par le polyethylene glycol. Apres fusion somatique, des jeunes plantes ont ete regenerees tandis que l'etape de regeneration a echoue dans les experimentations de transformation genetique

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36

Herbreteau-Lemonnier, Catherine. "Recherche dans un but de sélection des mécanismes impliqués dans la production de solasodine : utilisation de la variabilité observée in vitro (des cultures peu différenciées à la plante néoformée) chez Solanum laciniatum Ait. et Solanum khasianum C.B. Clarke". Paris 11, 1987. http://www.theses.fr/1987PA112446.

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Solanum laciniatum Ait. Et Solanum khasianum C. B. Clarke ont été utilisés pour établir des cultures de tissus et des clones néoformés à partir de ces cultures. Une méthode d'analyse chromatographique (chromatographie liquide sous haute pression) a été mise au point pour déterminer et quantifier les glycoalcaloïdes de la solasodine synthétisés par les différents tissus de ces Solanacées. La variabilité des cultures de tissus, suspensions de cellules et plantes néoformées sur cals a été mise en évidence pour des caractères morphologiques, physiologiques et biochimiques (contenu alcaloïdique et pigmentaire). Un modèle du développement, de la synthèse et de l'accumulation des alcaloïdes a pu être décrit chez cette espèce. Des caractères de sélection variétale ont été définis. L'hybridation somatique par électrofusion entre protoplastes de S. Khasianum et S. Melongena a été suivie de la régénération de plantes. L'une d'entre elles a été identifiée comme un hybride somatique après une étude morphologique et une analyse des isozymes. Solanum laciniatum Ait. And Solanum khasianum C. B. Clarke were used to establish tissue cultures and to regenerate plant clones. H. P. L. C. (High Pressure Liquid Chromatography) method for solasodine glycoalkaloids analysis was applied to our material. Morphological, physiological and biochemical (Glycoalkaloids and pigments content) variabilities were studies on callus suspensions and regenerated plants of S·laciniatum. A model of developmental stages of the plant and the pathway of alkaloid synthesis and accumulation can be defined in this species. Breeding characters are established. The somatic hybridization of S·khasianum and S·melongena by electrofusion of protoplasts permitted the regeneration of plants. One of the plants regenerated after the fusion was identified as a somatic hybrid by morphological and isozyme analysis
Solanum laciniatum Ait. And Solanum khasianum C. B. Clarke were used to establish tissue cultures and to regenerate plant clones. H. P. L. C. (High Pressure Liquid Chromatography) method for solasodine glycoalkaloïds analysis was applied to our material. Morphological, physiological and biochemical (Glycoalkaloïds and pigments content) variabilities were studied on callus suspensions and regenerated plants of S. Laciniatum. A model of developmental stages of the plant and the pathway of alkaloid synthesis and accumulation can be defined in this species. Breeding characters are established. The somatic hybridization of S. Khasianum and S. Melongena by electrofusion of protoplasts permitted the regeneration of plants. One of the plants regenerated after the fusion was identified as a somatic hybrid by morphological and isozyme analysis

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37

Simo, Santalla Pablo. "Contribution au développement de techniques pour l'hybridation somatique chez la pomme de terre, Solanum tuberosum culture et fusion de protoplastes de clones dihaploïdes, diploïdes hybrides interspécifiques et de l'espèce diploïde S. brevidens /". Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37618583d.

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38

Pensabene, Bellavia Giovanni. "Aplicación de la hibridación somática a la mejora de la citricultura española". Doctoral thesis, Universitat Politècnica de València, 2009. http://hdl.handle.net/10251/6361.

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Abstract (sommario):

La hibridación somática, o fusión de protoplastos, es una herramienta biotecnológica aplicable a la mejora de los cítricos. La utilización de esta técnica permite abordar objetivos que sería imposible plantear mediante la hibridación sexual u otras técnicas de mejora, tanto a nivel de mejora de variedades y patrones ya existentes, como para la obtención de nuevos genotipos de interés. El objetivo principal de esta tesis ha sido la aplicación de la hibridación somática a necesidades específicas de la citricultura española. Además se han realizado estudios sobre aspectos técnicos relacionados con la hibridación somática, con el objetivo de facilitar el trabajo de mejora que se realiza aplicando esta técnica. La tesis ha sido dividida en cuatro capítulos y dos apéndices que se describen a continuación. El primer capitulo está dirigido hacia la obtención de nuevos patrones de interés utilizando genotipos con características complementarias. Para ello se escogieron los genotipos citrange "Carrizo" y Citrus macrophylla que son dos de los patrones más utilizados en el cultivo de los agrios. Se compararon directamente la eficacia de los tres métodos de fusión de protoplastos disponibles para cítricos, aplicándolos paralelamente a un mismo cruzamiento. En el segundo capítulo se muestra la caracterización genética exhaustiva de las plantas obtenidas, tanto a nivel nuclear como a nivel citoplasmático. Los resultados de esta caracterización mostraron que los híbridos somáticos no siempre son la suma perfecta de los dos parentales y que puede haber cambios a nivel cromosómico, subcromosómico o citoplasmático. El tercer capítulo muestra el estudio llevado a cabo del efecto de la radiación con luz UV sobre la integridad del genoma de los protoplastos de cítricos. El interés en la aplicación de este tipo de radiación reside en la posibilidad de obtener híbridos somáticos asimétricos.
Pensabene Bellavia, G. (2009). Aplicación de la hibridación somática a la mejora de la citricultura española [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/6361
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39

Pavan, Alexandra. "Fusão de protoplastos de citros e avaliação da resistência do híbrido somático laranja 'Hamlin' + mexerica 'Montenegrina' a Xanthomonas axonopodis pv. citri e Xylella fastidiosa". Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/11/11144/tde-03102006-164336/.

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Buscou-se produzir híbridos somáticos entre laranjas doces (Citrus sinensis) com tangerinas, mexericas (C. reticulata, C. reshni, C. sunki, C. deliciosa), tangores (C reticulata x C. sinensis) ou tangelo Orlando (C. reticulata x C. paradisi), além da avaliação da resistência do híbrido somático laranja Hamlin (C. sinensis) + mexerica Montenegina (C. deliciosa) a Xanthomonas axonopodis cv. citri e Xylella fastidiosa. Foram utilizados como fonte de protoplastos calos embriogênicos e folhas coletadas de plântulas germinadas in vitro e de plantas cultivadas em casa-de-vegetação. Após o isolamento, protoplastos provenientes de calos e/ou suspensões embriogênicas foram fundidos quimicamente por polietilenoglicol (PEG) com protoplastos não embriogênicos oriundos de mesofilo foliar. Os calos provenientes da fusão foram induzidos a formação de embriões somáticos para posterior germinação e regeneração de plantas. As plantas regeneradas foram individualizadas, enraizadas ou microenxertadas e aclimatizadas em casa-de-vegetação. A confirmação da hibridação somática foi feita por análise morfológica, análise do DNA com marcadores moleculares do tipo RAPD, determinação da ploidia com a contagem do número de cromossomos e/ou citometria de fluxo. Foram obtidos dois híbridos somáticos do genitor embriogênico laranja Hamlin com os genitores não embriogênicos mexerica Montenegrina e tangerina Dancy. Ambos híbridos somáticos obtidos podem apresentar características complementares dos genitores, podendo ser utilizados diretamente como copa e em programas de melhoramento de copa de citros. O híbrido somático laranja Hamlin com mexerica Montenegrina foi avaliado mostrando resistência a Xanthomonas axonopodis cv. citri e a Xylella fastidiosa.
This research aimed to produce new somatic hybrid combinations between sweet orange (Citrus sinensis) with mandarins (C. reticulata, C. reshni, C. Sunki, C. deliciosa), tangors (C reticulata x C. sinensis) or Orlando tangelo (C. reticulata x C. paradisi), and also evaluate the somatic hybrid Hamlin sweet orange (Citrus sinensis) + Montenegrina mandarin (Citrus deliciosa) for resistance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa. Protoplast sources included embryogenic calli or suspension-culture derived calli, and leaves collected from plants cultivated in vitro or in screenhouses. After protoplast isolation, embryogenic protoplasts were chemically fused by polyethylene glycol (PEG) with mesophyll-derived non-embriogenic protoplasts. Fusion-derived calli were further cultured to embryo induction, germination, and plant regeneration. Regenerated plants were individually rooted or micrografted, and further acclimated in screenhouse. Somatic hybridization was confirmed by analysis of leaf morphology, molecular analysis by RAPD markers, ploidy determination by chromosome counting or flow cytometry. The producion of the somatic hybrids Hamlin sweet orange + Montenegrina mandarin and Hamlin sweet orange + Dancy mandarin was confirmed. These hybrids may have complementary traits from both progenitor and be used directly as scion cultivars or as parental lines in scion improvement programs. Hamlin sweet orange + Montenegrina mandarin somatic hybrid was resistant to Xanthomonas axonopodis pv. citri and Xylella fastidiosa.

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YANG, SHIOW-JING, e 楊秀菁. "Protoplast fusion of Ganoderma lucidum and G. tsugae". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/36498120360981215828.

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LU, MEI-HUA, e 盧梅華. "Protoplast fusion of L-Lysine producers from Brevibacterium divaricatum". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/59311712039899433200.

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42

"Study on the interspecific hybridization of pleurotus by protoplast fusion". Chinese University of Hong Kong, 1985. http://library.cuhk.edu.hk/record=b5885585.

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43

Zhang, Wen Hui, e 張文慧. "Studies on protoplast fusion and fermentation process of mycobacterium sp". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/51933823749778942715.

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44

"Intergeneric hybridization of schizophyllum commune and pleurotus florida by protoplast fusion". Chinese University of Hong Kong, 1993. http://library.cuhk.edu.hk/record=b5887849.

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Abstract (sommario):

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

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45

"Isolation, identification and application of protoplast fusion products in edible mushrooms". Chinese University of Hong Kong, 1994. http://library.cuhk.edu.hk/record=b5888202.

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Abstract (sommario):

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

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46

Chen, Liang-Yun, e 陳良雲. "Protoplast fusion of Trichoderma koningii and Trichoderma harzianum as a biocontrol agent". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/39633469244015805569.

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Abstract (sommario):

碩士
國立中興大學
植物學系
82
The objective of this paper was to set up the protoplast fusion technique for Trichoderma spp.,to obtain the stable and high frequency fusants,and to improve biocontrol effect.At first, pSV50 with anti-bonomyl gene was transfered to T.koningii and stable transformants were selected. Two kinds of protoplasts were fused by combining the selected anti-hygromycin B transformant from T. harzianum and the anti-benomyl transformant. Protoplasts from two transformants of T. koningii and T. harzianum, obtained from young thalli following cell wall digestion by Novozym 234, were fused in final 33% PEG suspended in 10mM Tris-HCl and 10mM CaCl2, pH 7.5. The frequency of fusion between anti-hygromycin B and anti-benomyl transformants was about 2-5% .The ability of all fusants, transformants and wild type to overgrow the soil-borne pathogenic fungi Rhizoctonia solani in dual culture was used to evaluate their antagonistic capability. The antagonistic ability of the fusant strains were found to vary with pathogenic fungi. The fusants were improved for their antagonistic ability by genetic purification (subculture).By statistical analysis,we could find that fuants and transformants had no distinct difference from parental strains. As to the effect of antibiotics on growth ,it exhibited some varities in fusants and fusants were similiar with parental strains. The results of DNA content and segregation analysis indicted that the interspecific funants had no segreation ,homologous, and haploid. DNA-DNA Southern hybridization revealed the integration of the pUCH1 and pSV50 plasmids into the fusant chromosome DNA .It indicated that a sucessful fusion was no longer a mutation.

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47

ZHOU, LONG-WU, e 周隆武. "Studies on protoplast fusion of cholesterol oxidase-producings trains of Arthrobacter simplex". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/11198610699771161311.

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48

HUANG, JIAN-XIONG, e 黃健雄. "Studies on corynebacterium protoplast fusion technique and the preparation of a BOD sensor". Thesis, 1989. http://ndltd.ncl.edu.tw/handle/25600174610949090803.

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49

NI, DE-GUAN, e 倪德全. "Studies on the properties and breeding by protoplast fusion of saccharomyces cerevisiae shaohsing yeasts". Thesis, 1990. http://ndltd.ncl.edu.tw/handle/43319258177165135427.

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50

ZHENG, CHENG-HAN, e 鄭誠漢. "Protoplast fusion of rice (Oryza sativa L.) and barnyard grass (Echinochloa crus-galli Beauv. var formosaensis Ohwi)". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/55559990918603899773.

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