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1
Cheng, Jianping. "Inheritance of protoplast culturability and improvement in pollen development by protoplast manipulation in solanum." Diss., This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-09162005-115010/.
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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.
3
Wilson, J. M. "Protoplast-mediated genetic manipulation of brewing yeasts." Thesis, University of Nottingham, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355426.
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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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Dunn, R. M. "The protoplast mediated genetics of thermophilic bacilli." Thesis, Cardiff University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380254.
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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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Seear, Paul James. "Protoplast production and interspecific hybridisation in Lupinus albus." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326751.
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8
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.
9
Attree, S. M. "Properties of Pteridium protoplasts." Thesis, University of Manchester, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378009.
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Thompson, J. A. "Plant regeneration from cell and protoplast cultures of rice." Thesis, University of Nottingham, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378368.
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Tamura, Mihoko. "Ploidy Manipulation through Protoplast Culture Techniques for Persimmon Breeding." Kyoto University, 1997. http://hdl.handle.net/2433/202415.
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Khehra, Gurpreet Singh. "Genetic manipulation in rice using rice tungro spherical virus coat protein genes." Thesis, University of Nottingham, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261168.
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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
Graduate
14
Finch, Robert P. "Development of protoplast systems for the genetic manipulation of rice." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280099.
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Baset, Abdul. "Protoplast to plant regeneration systems for cultivated and wild rices." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315069.
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Ghadimzadeh, Mortaza. "Studies of protoplast and liposome techniques in plant tissue culture." Thesis, University of Salford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.255239.
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Foulger, David. "Protoplast regeneration and somatic hybridisation of potato (Solanum tuberosum L.)." Thesis, Rothamsted Research, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374242.
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Butt, Adrian David. "Physiological and biochemical studies with rubber (Hevea brasiliensis) protoplasts." Thesis, University of Nottingham, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257197.
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Nobre, Jose Manso Preto. "Studies on methods for the genetic manipulation of barley (Hordeum vulgare L.)." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336932.
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Wardrop, Julie. "Biotechnological applications of perfluorochemical liquids in plant tissue culture." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389475.
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Chen, W. H. "Genetic manipulation of sugarcane." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376164.
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Cui, Xiucheng. "Targeted Gene Editing Using CRISPR/Cas9 in a Wheat Protoplast System." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36543.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a promising tool for targeted gene editing in a variety of organisms including plants. In this system, a 20 nt sequence on a single guide RNA (sgRNA) is the only gene-specific information required to modify a target gene. Fusarium head blight (FHB) is a devastating disease in wheat caused by the fungus Fusarium graminearum. The trichothecene it produces, deoxynivalenol (DON), is a major mycotoxin contaminant causing food production loss both in quality and yield. In this project, we used the CRISPR/Cas9 system to modify three wheat genes identified in previous experiments, including an ABC transporter (TaABCC6), and the Nuclear Transcription Factor X box-binding-Like 1 (TaNFXL1), both associated with FHB susceptibility, and a non-specific Lipid Transfer Protein (nsLTP) named TansLTP9.4 which correlates with FHB resistance. Two sgRNAs were designed to target each gene and were shown in an in vitro CRISPR/Cas9 assay to guide the sequence-specific cleavage with high efficiency. Another assay for CRISPR/Cas9 was established by the optimization of a wheat protoplast isolation and transformation system. Using a construct expressing a green fluorescent protein (GFP) as a positive control, estimated transformation efficiencies of about 60% were obtained with different batches of protoplasts. High-throughput sequencing of PCR amplicons from protoplasts transformed with editing constructs clearly showed that the three genes have been successfully edited with efficiencies of up to 42.2%. In addition, we also characterized by RT-qPCR the expression pattern of 10 genes in DON-treated protoplasts; seven of the genes were induced by DON in the protoplasts, consistent with their previously identified DON induction in treated wheat heads, while three genes expressed differentially between DON-treated wheat heads and protoplasts. Preliminary bioinformatics analyses showed that these differentially expressed genes are involved in different plant defense mechanisms.
23
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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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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Redway, F. A. "Regeneration in tissue and protoplast culture of Sainpaulia ionantha (African violet)." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382673.
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Slamet, Inez Hortense. "Protoplast culture and somatic hydridisation of Indica and Japonica rice cultivars." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335365.
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Aziz, Zaleha Biniti A. "Tissue culture of Centella asiatica : asiaticoside biosynthesis." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368364.
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Tang, Kexuan. "Studies on rice protoplast culture and transformation using an insect resistance gene." Thesis, University of Nottingham, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294301.
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Barakat, M. N. R. "Protoplast and tissue culture studies for somatic hybridisation in the genus Linum." Thesis, University of Nottingham, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304777.
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Gilbert, Julie E. "Interaction between mixing and density in protoplast cultures of Solanum tuberosum L." Thesis, University of Reading, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314683.
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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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Costa, Natalia Layane Badaró. "Improved procedures to assess plant protoplast viability: evidencing cytological and genomic damages." Universidade Federal de Viçosa, 2017. http://www.locus.ufv.br/handle/123456789/11949.
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Em todas as aplicações, o teste de viabilidade e necessario para medir a taxa de protoplastos viáveis, possibiltando decidir os procedimentos de isolamento e purificação mais adequados e verificar se ha celulas suficientes para as etapas subsequentes. A microscopia de fluorescência geralmente é empregada para o teste de viabilidade. No entanto, alguns problemas têm sido apontados: longo tempo necessário para contar um número relativamente pequeno de protoplastos, aglomerados de células que impedem a observação dos protoplastos e percepção visual da fluorescência subjetiva ao observador. Este estudo teve como objetivo estabelecer procedimentos para teste de viabilidade adaptado para citometria de fluxo (FCM), MuseTM cell analyzer (Muse) e Ensaio cometa (CA). Para isso, Capsicum annuum foi escolhido devido a natureza morfogênica recalcitrante dos protoplastos. Após o isolamento e a purificação, as aplicações permitiram avaliar um grande número de protoplastos (FCM e MUSE) e núcleos dos protoplastos (CA) em um curto período de tempo. A partir das adaptações nos procedimentos, foram evidenciados diferentes tipos e níveis de danos citológicos (FCM e Muse) e genômicos (Muse e CA), possibilitando discriminar e mensurar os protoplastos viãveis. Considerando os resultados, este estudo introduz procedimentos quantitativos melhorados para o teste de viabilidade. Alem disso, visando a regeneração de plântulas, diferentes métodos podem ser aplicados para avaliar a viabilidade de protoplastos, definindo os procedimentos de isolamento e purificação mais adequados. Corraborando para este propósito, foram mostrados guias para FCM, Muse e CA visando a padronizaçao dos testes de viabilidade em protoplastos de plantas.
Plant protoplasts are valuable in biotechnology, enabling the plantlet regeneration until the gene function determination. In all applications, viability test is required to measure the rate of viable protoplasts, allowing to decide on the most adequate isolation and purification procedures and to verify whether there are sufficient cells for subsequent steps. Fluorescence microscopy is usually employed for viability test. However, some problems have been pointed out: long time required to count a relatively small number of protoplasts, cell clumps preventing their observation, and the subjective visual perception of the fluorescence by observer. This study aimed to establish procedures for viability test adapted for flow cytometry (FCM), MuseTM cell analyzer (Muse) and Comet Assay (CA). For this, Capsicum annuum was chosen due to recalcitrant morphogenic nature of its protoplasts. After isolation and purification, the applications allowed assessing large numbers of protoplasts (FCM and MUSE) and protoplasts nuclei (CA) in a short time period. From the adjusted procedures, different types and levels of cytological (FCM and Muse) and genomic damages (Muse and CA) were evidenced, allowing to discriminate and measure the viable protoplasts. Considering the results, this study introduces improved quantitative procedures for viability test. Besides of these and aiming the plantlet regeneration, different methods can be applied to assess the protoplast viability, defining the more adequate isolation and purification procedures. Contributing with this purpose, guides were showed for FCM, Muse and CA to standardization of viability tests in plant protoplasts
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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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Qusus, Saba J. "Molecular Studies on Soybean Mosaic Virus-Soybean Interations." Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/30328.
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In the U.S., soybean mosaic virus (SMV) is classified into seven strain groups, designated G1 to G7, based on their different responses on resistant soybean [Glycine max (L.) Merr.] cultivars. These responses are: symptomless or resistant (R), necrotic (N), and mosaic or susceptible (S). The gene-for-gene model has been proposed for SMV-soybean interactions. In the majority of cultivars, a single dominant gene, Rsv1, confers both the R and N responses. In the first part of this study, the coat protein (CP) genes of two SMV strains, G1 and G6 were isolated, cloned, and sequenced. Gene isolation was done by reverse transcription-polymerase chain reaction (RT-PCR) on partially purified virus preparation without prior RNA extraction. Amplified products were blunt-end ligated into pNoTA/T7 vector and transformed into competent cells. Sequencing was performed in both directions on heat-denatured double-stranded plasmids. The predicted 265 amino acid sequence of the CP of G1 and G6 strains were 98.9% identical, with only two amino acid differences. Correlating the CP sequences of G1, G2, G6, and G7, with their virulence on resistant soybean cultivars indicated that the CP is not likely to be the R- and/or N-determinant in the SMV-soybean system. The second part of the study involved studying the pathogenesis of G1, G6, and G7 strains on inoculated leaves of R, N, and S soybean cultivars by leaf imprint immunoassay. Results indicated four types of reactions: i) susceptible, showing unrestricted replication and spread; ii) immune, where no virus was detected; iii) systemic spread, showing unrestricted replication but limited spread along the veins; and iv) restricted replication and spread, where infection was restricted to few foci along the veins. Results of this study indicated that Rsv1-mediated resistance is a multicomponent type of resistance that involves both inhibition of virus replication as well as cell-to-cell movement. The third part of the study aimed at investigating Rsv1-mediated resistance at the cellular level. For this purpose, an SMV-soybean protoplast system was developed. Protoplast isolation was based on a combined cellulase-pectolyase Y-23 digestion and metrizamide-sorbitol gradient purification protocol. Virus inoculation of protoplasts was facilitated by either polyethelene glycol (PEG) or poly-L-ornithine (PLO), and method of detection was by Western blotting using antiserum to whole virus. Inoculation by PEG was successful, but results were irreproducible because of the adverse effect of PEG on protoplast viability. Inoculation by PLO was inconclusive because of the high background from residual inoculum. Additional research is needed before a protoplast system can be used to study the mechanism of Rsv1 resistance to SMV at the cellular level.Ph. D.
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Castro, Lívia Mendes de. "Isolamento, cultura de protoplastos e regeneração de plantas de laranja doce (Citrus sinensis L. Osbeck)." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/11/11136/tde-16032010-164451/.
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A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin.Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.
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Nahar, Maksuda Anjuman. "Regeneration of plants from anther, callus and protoplast cultures of rice (Oryza sativa L.)." Thesis, University of Nottingham, 1994. http://eprints.nottingham.ac.uk/13505/.
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The work described in this thesis focuses on the development of fully reproducible systems of plant regeneration through embryogenic callus and totipotent protoplast cultures of rice (Oryza sativa L. cv. Taipei 309). The calli originated from different explants, including excised anthers, isolated microspores, mature seed-embryos, immature seed-embryos and leaf-base meristems. In the callus culture and plant regeneration studies, excised anther-derived callus produced higher numbers of green (90.5%) and albino (9.5%) plants, both haploids (81.2%) and diploids (18.8%), compared to other explants. The plant regeneration frequency from mature seedembryo-derived callus varied with callus age and embryogenicity of the callus. A maximum of 20% plant regeneration was obtained with approx. 2% of the regenerated plants being albinos. In the case of mature or immature seed-embryos and leaf-base meristems, plant regeneration frequency from callus was dependent on some more specific factors, in addition to callus age and embryogenicity. These factors were the age of seedlings grown in vitro and the age of immature embryos after anthesis for leaf-base meristem and immature embryo cultures, and also the scutellar surface orientation of mature or immature embryos during callus culture initiation. The present study also focused on the plating efficiencies and plant regeneration frequencies obtained from culture of embryogenic cell suspension-derived protoplasts. These varied with the explant source, the embryogenic nature and age of the callus during suspension initiation, the age of the embryogenic suspension culture, the time of enzymatic digestion, the plating density, viability of protoplasts and use of the suspensions at their exponential (actively dividing) growth stage. The results from this study indicated that leaf-base meristem and anther calli-derived suspensions retain their regenerability for a longer time compared to suspensions derived from mature seedscutellum and immature seed-derived embryos. Maximum plant regeneration frequency (45%) was obtained from embryogenic suspensions derived from mature seedembryos. A detailed study of different agronomic characteristics of 57 protoclonal plants (mature seed-scutellum-derived) was performed and the statistical analysis of the data indicated a range of variability among the protoclonal plants and also between the protoclonal and seed-derived plants. A detailed study of the ploidy levels of regenerated plants obtained through different culture systems (callus and protoplasts) was performed by flow cytometry and chromosome counting of meristematic cells. The results showed that anther calliderived plants were mostly haploids, protoplast-derived plants of leaf-base meristem, anther, and mature seed-scutellum origin were mostly tetraploids and in between these two groups, diploids, triploids and aneuploids were also present.
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Ayeleso, Taiwo Betty. "Protoplast isolation and plant regeneration in Bambara groundnut : a platform for transient gene expression." Thesis, Cape Peninisula University of Technology, 2016. http://hdl.handle.net/20.500.11838/2003.
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Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016.Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.
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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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Sinha, Debleena. "Development of an In Vitro Protoplast Culture System for Albizia Lebek (L.) Benth., an Economically Important Leguminous Tree." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc500422/.
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An in vitro system of generating protoplasts from their callus cultures was established. The friable callus was more productive in terms of producing protoplasts than the green compact callus. The concentration of the various cell wall degrading enzymes had an effect on the viability of the protoplasts in the medium. The protoplast system developed from the experiments was stable and could be used for the transformation experiments of Albizia lebek and for other plant improvement practices.
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Mujahid, Hana, Feng Tan, Jian Zhang, Babi Ramesh Nallamilli, Ken Pendarvis, and Zhaohua Peng. "Nuclear proteome response to cell wall removal in rice (Oryza sativa)." BioMed Central, 2013. http://hdl.handle.net/10150/610238.
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Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.
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Daneri, Castro Sergio Nicolas. "Germination-related cell death in the aleurone layer of malting barley." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15893.
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Malting of barley grain represents the most economically favourable application of this cereal. Most studies of barley grain germination have stemmed from the need to optimise the malting process, thus saving resources and obtaining a superior product. The need to optimise the malting process remains important and represents the background for the research reported in this thesis. One of the most functionally important tissues of the barley grain during germination is the aleurone layer, since it secretes most of the hydrolytic enzymes necessary for the degradation of the products stored in the endosperm. The extent of the degradation of complex carbohydrates and proteins of the endosperm is a crucial parameter evaluated during the malting process. The aleurone layer cells use their own reserves for the production and secretion of the hydrolytic enzymes, and once these have been depleted the cells die under a programmed cell death response. This thesis describes the development of two techniques to study programmed cell death of aleurone layer cells of malting barley varieties under conditions that replicate germination. The first technique developed was a protocol for the isolation of aleurone-layer protoplasts from mature dry grains. The isolated protoplasts were shown to be viable and were successfully characterised to evaluate their death response via incubation with gibberellic acid (GA). The second technique developed was the use of activity-based protein profiling (ABPP) for the characterisation of enzyme activities in aleurone layer cells. Through the use of ABPP, changes in activity of various groups of enzymes were evaluated for isolated aleurone layers and aleurone-layer protoplasts upon incubation with GA in the presence of abscisic acid (ABA). The techniques developed in this thesis will improve the understanding of the behaviour of the aleurone layer during germination. This knowledge is likely to contribute to the optimisation of the barley malting process.
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Mat, Yunus Abdul Masani [Verfasser]. "Development of a protoplast-based transformation system for genetic engineering of oil palm / Abdul Masani Mat Yunus." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2013. http://d-nb.info/1037936132/34.
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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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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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Lee, Sung-Ho. "Genetic transformation of rice via a protoplast-to-plant regeneration system using the rolA gene from Agrobacterium rhizogenes." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307761.
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Jelodar, Nadali Babaeian. "Protoplast culture and somatic hybridisation of cultivated rice, Oryza sativa, and the wild relative of rice, Porteresia coarctata." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321371.
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Taylor, Thomas E. "Inheritance of competencies for leaf disc regeneration, anther culture, and protoplast culture in Solanum phureja and correlations among them." Thesis, This resource online, 1991. http://scholar.lib.vt.edu/theses/available/etd-10242009-020252/.
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Le, Minh Phuong. "Biotransfer of selected risk metals into plants and their accumulation and distribution in plant organs." Doctoral thesis, Česká zemědělská univerzita v Praze, 2016. http://www.nusl.cz/ntk/nusl-259725.
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Contamination of soils with heavy metals is one of the serious environmental problems threatening human being. Heavy metals are considered as the special hazard of soil pollutants because of the adverse effects on the plant growth, the amount, activity of useful microorganisms in soils and the quality of food. Regard to the persistent and toxicity, the heavy metals are toxic when we consider different kinds of pollutants in soils. In the soil, zinc (Zn), cadmium (Cd), lead (Pb) and mercury (Hg) toxicities frequently occur than the other metals because of their precipitation and sorption by the soil. It is a very dangerous situation because when these metals are taken up by plants, they can be transported to the food web and food chains.
In the present study, the accumulation of four heavy metals (mercury, zinc, lead and cadmium) in the whole grain of spring accessions of emmer, einkorn and common spring wheat cultivars and potato (Solanum tuberosum) is reported. Heavy and essential elements were monitored in potato cultivars in the exact field experiments and in hydroponically grown plants. The elements were determined by methods FAAS, ET AAS, and AMA (Advance Mercury Analysis). Statistical analyses were performed using SPSS 9.0 with the Tukey HSD (Honestly Significant Difference) test (alpha equal to 0.05).
In our study, the concentration of heavy metals decreased in the order zinc (Zn) > lead (Pb) > cadmium (Cd) > mercury (Hg) in the wheat grain. The comparison between three varieties of investigated wheat revealed that the emmer wheat was rich in zinc content (62.12 mg kg-1 dry matter), while the spring wheat had the lowest average concentration of zinc in the grain (40.99 mg kg-1 dry matter). Generally, the values of lead concentration in grain wheat varieties were low (ranging from 0.1268 mg kg-1 dry matter to 0.2950 mg kg-1 dry matter).
The concentrations of mercury in four typical growth stages of wheat (boot stage 10, heading stage 10.2 1/4 of head emerged, leaf-stage 10.2 and stage ripening 11 according to Feekes) were also determined. It has been shown that the concentrations of mercury in different wheat varieties were absorbed differently at different growth stages of plant. Stage 10.2 and leaf stage 10.2 showed the high mercury content (0.0152 mg kg-1 dry matter and 0.0214 mg kg-1 dry matter, respectively). Among individual varieties significant differences were determined.
Amounts of toxic and potentially toxic elements detected in investigated potato tubers are characterized by a large variability within investigated groups. Performing statistical analysis (one way ANOVA) showed that there were no significant differences between two investigated groups of samples (samples from Uhříněves and Valečov in the year 2013 and 2014) considering either one of investigated metals. Measurable levels of mercury were found in smallest amounts in all investigated potato samples comparing to other metals (Cd, Pb).
Plant cells compared to animal cells are characterized by the formation of cell walls. Plasma membrane or cell membrane is a biological active membrane separating the interior of cell from the outside environment. An adjusted method for isolation of protoplasts was developed and adapted for isolation of protoplasts from plant material (potatoes). In our experiment, the plants were grown hydroponically in the Research Institute of Plant Crops Prague-Ruzyně. If we examine the plant membrane, one option is to remove the cell wall by means of special mixture enzymes. Protoplasts were released in the dark at 25 degrees of Celsius for 18 hours. The 70 and 90 microns sieve was used to filter and then centrifugation for 5 minutes at 100 x g. All the steps were carefully carried out to prevent the damage or breakage of protoplasts.
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WANG, MIN-XING, and 王敏行. "Observation on the suspension cell and protoplast cultures and wall regeneration from protoplasts of daucus carota L." Thesis, 1988. http://ndltd.ncl.edu.tw/handle/54521976442766643631.
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林道一. "The Isolation and Culture of Phalaenopsis Protoplast." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/30108065759306429253.
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碩士東海大學
生物學研究所
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There are very few papers reported on isolation and culture oforchid. Besides, no plant has ever been regenerated from therchid. It may due to that there is a large number of crystalhe orchid tissue which reduce the yield of protoplasts. Severalen tried to overcome the effect of crystals on protopalsto increase the yield of protoplasts in Phalaenopsis. The crystalsn 0.2 M EDTA or 2 N HCl in vitro. However, the reagents will both damage the protoplasts.n not be broken down by Rowatinex and ultrasonic wave treatments.solution can separate the intact protoplasts from crystals andng protoplast isolation. Phalaenopsis seedlings cultured inrient deficient media before mesophyll protoplasts isolation willlast yield. However, dark culture before protoplasts isolationhe yield of protoplast. Incubate Phalaenopsis leaves in solution containing 1% Cellulased 1% or 0.2% Macerozyme R-10 for 4 to 6 hours will yield 0.3 - 3.. It would have higher protoplast yield after 15 day'srotocorm (5.86 X 106 pps/g fw.). The isolated protoplasts werell wall synthesis when cultured in KM8P, PM1, LBP, and N6P media.st medium in which protoplasts were able to divide twice, an not form colony and die after 2 to 3 weeks of culture. The "budding"lts from either the somatic fusion of two adjacent protoplasts orbudding of a single cell. Incubate budding cells in 0.8%ion will produce spherical protoplasts indicating that celluloseg culture. Addition of DMSO to medium does not inhibit budding ofas the concentration of DMSO increase the viability of theease.