Tesi: "Protoplasts"

1

Lynch, Paul Thomas. "The uptake of fungal protoplasts by plant protoplasts". Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328131.

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2

Mullins, P. J. "Protoplasts from Phytophthora". Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381366.

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3

Lawrence, W. A. "Microinjection of tobacco protoplasts". Thesis, University of East Anglia, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372559.

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4

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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5

Sheard, J. P. "Glucose uptake by pea mesophyll protoplasts". Thesis, University of East Anglia, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235210.

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6

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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7

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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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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8

Bourne, N. "The regeneration and transformation of Bacillus protoplasts". Thesis, Cardiff University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376554.

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9

Newell, Jane Marie. "Vacuole development in evacuolated oat mesophyll protoplasts". Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295919.

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10

Ward, Kenneth Glenn. "Microinjection and regeneration of tobacco and potato protoplasts". Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303971.

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11

Doulis, Andreas G. "Antioxidant responses of pea (Pisum sativum L.) protoplasts". Diss., This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-09192008-063125/.

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12

Kuroki, Mayra Akemi. "Desenvolvimento de uma estratégia de clonagem customizada de regiões promotoras do genoma da cana-de-açúcar". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-05022013-082130/.

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O objetivo deste trabalho foi desenvolver uma metodologia para identificação de regiões promotoras funcionais a partir de segmentos de um genoma qualquer. O genoma da cana-de-açúcar foi escolhido para o desenvolvimento desta estratégia na qual envolve a obtenção de fragmentos de DNA os quais foram clonados em vetores de expressão. A triagem destes fragmentos foi realizada através de biobalística e resultou no isolamento de quatro clones. Um ensaio de transformação permanente em arroz com três clones gerou 12 plantas. Foi detectada expressão do marcador GUS em calos, folhas e raízes, comprovando sua funcionalidade. Desta maneira, o presente trabalho permitiu estabelecer uma metodologia de recuperação de sequências regulatórias funcionais com ampla possibilidade de serem explorados biotecnologicamente.
The aim of this work is develop a strategy to identify functional promoter regions from any genome. The modern sugarcane genome was chosen as a model for the development of this strategy that involves the generation of fragments of DNA and cloning them into expression vectors. These fragments were then screened by a transient expression assay using biolistic particle delivery resulting in the isolation of four clones. Three clones were permanently transformed in rice, and 12 plants were obtained. GUS expression was detected in the callus, leaves and roots of the rice plants thus confirming the functionality of sequences in these clones. The present work has established a strategy to identify and extract functional regulatory sequences containing functional regulatory regions which show great potential of being useful in both the biotechnology field and in the field of basic science.

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13

Hörtensteiner, Stefan. "Re-formation of vacuoles in evacuolated tobacco mesophyll protoplasts /". [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10426.

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14

De, Both Michiel Theodoor Jan. "Gene transfer by electroporation in plant protoplasts and tissues". Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46278.

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15

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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16

Sproule, Anne Carleton University Dissertation Biology. "Plant regeneration from stem cortex protoplasts of Brassica juncea". Ottawa, 1987.

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17

Crotty, Christopher M. "Two distinct outward K+ conductances are simultaneously activated in TBY-2 suspension culture protoplasts". Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38174.

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Two kinetically and pharmacologically distinct outward K+ conductances were found to be simultaneously activated at the plasma membrane of TBY-2 suspension culture protoplasts using the whole-cell patch-clamp technique. We used a modified Hodgkin-Huxley model to quantify the activation kinetics of the outward current. Time constants for the two conductances derived from the model differed in magnitude by 5--10 fold over the voltage range from -10 mV to +50 mV, allowing their classification as either fast or slow. Deactivation kinetics were better fit by two exponential terms rather than one, yielding fast and slow deactivation time constants. The voltage dependence of time constants derived from these two independent two-channel models followed a bell-shaped distribution with mid-point potentials for both components at -20 mV with a standard 10-fold K+ gradient (10 K+o/100K+i).
Both components were highly K+-selective, however the tail current amplitudes of the slowly activating component at hyperpolarized potentials exhibited non-linear rectification whereas the tail current amplitudes of the fast activating component were linear. The ratio of inward tail current/activated outward current (envelope of tails test) was not constant during the depolarizing step; during the first 50--100 milliseconds the ratio was 6 times higher than at quasi-steady-state (i.e. after 0.3 second).
A pharmacological dissection of outward currents revealed that external Ba2+ in the range from 10 muM to 1 mM selectively inhibited a fast, sigmoidally activating, slowly inactivating current as revealed by examining difference currents. The more slowly activating component was inhibited by only 20% with 5 mM Ba2+. Conversely, nitrendipine or bepridil (5--100 muM) selectively inhibited the slower component of outward current. External TEA inhibited both the fast and slow components equally; tail current amplitudes of both components were inhibited by 40% with 2 mM TEA and the activation time courses in the presence of TEA conserved the same kinetic parameters as control currents.

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18

Foreman, Julia Louise. "Genetic and molecular analysis of root hair initiation and tip growth in Arabidopsis thaliana". Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247111.

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19

Lamb, Mary Patricia. "Osmolality tolerance and ion channels in protoplasts of entomophthoralean fungi". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0006/MQ34198.pdf.

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20

Spice, Nicola Jane. "Evaluating the use of fungal protoplasts to investigate fungicide action". Thesis, Nottingham Trent University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341273.

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21

Curtis, Nicola. "Protoplasts as tools for the genetic study of Agraricus bisporus". Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335201.

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22

George, Alexander Christopher Stephen. "Magnetofection of tobacco protoplasts: a novel mechanism for plant transformation". Thesis, George, Alexander Christopher Stephen (2018) Magnetofection of tobacco protoplasts: a novel mechanism for plant transformation. Honours thesis, Murdoch University, 2018. https://researchrepository.murdoch.edu.au/id/eprint/49707/.

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To help meet the challenges of climate change and a growing global population, it has never been more critical that the yields of agricultural crops are increased: new breeding technologies such as genetic engineering can play a major role in achieving increased food production. Despite several decades of study there are still limitations to plant transformation technologies. These include transformation methods based on vector-mediated introduction of gene constructs using Agrobacterium, or direct gene transfer methods such as particle bombardment or transformation of protoplasts using polyethylene glycol or other methods to disturb cell membranes. Such limitations have slowed applications to generate genetically engineered plants with improved properties. One potentially new approach to transform cells is based on nanoparticle-mediated transformation. The use of nanoparticles as gene vectors may overcome some current limitations when applied to plant systems. The small size and novel properties of nanoparticles can be exploited since the physicochemical properties of nanoparticles can be modified to suit different applications, so that they can deliver a diverse range of biomolecules. One such novel system is termed ‘magnetofection’, where superparamagnetic iron oxide nanoparticles coated with biomolecules (magnetofectins) are directed towards target cells by application of a strong external magnetic field. Magnetofection has been studied for almost two decades in animal systems, but to date there are only two reports of magnetofection being applied to plant cells. This study was a ‘proof-of-concept’ to determine whether magnetofection could be used as a transformation system for tobacco mesophyll protoplasts, including optimisation of conditions, and to study the effects of magnetofection treatments on the viability of protoplasts. In initial experiments, a transgenic line of cotton was obtained which expressed the reporter gene β-glucuronidase (GUS), and systems were developed to assay GUS expression both by histochemical staining and fluorescence. A GUS expression plasmid (pCAMBIA1303) was obtained for experimental work and its identity checked. pCAMBIA1303 was bound via electrostatic interactions to PEI derivatised 100nm superparamagnetic iron oxide nanoparticles. After application of a powerful external magnetic field, the magnetofectins were attracted towards protoplasts, and transient gene expression of GUS was demonstrated 48 hours post transfection. Of the tested complexes, the transformation efficiency was highest when magnetofectins were assembled with 1:5 and 1:10 MNP:DNA weight ratios. It was shown that the density of MNPs affected the viability of magnetofected protoplasts, and that increasing MNP density significantly reduced protoplast viability. An optimal density of MNPs (1.0μg/mL) was found that resulted in transient GUS expression and did not affect protoplast viability. This work provides a starting point for further development of magnetofection as a novel plant transformation system, the advancement of which may broaden the scope of plant molecular biology and genetic engineering.

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23

Saka, Kamel. "REGENERATION OF COTTON (GOSSYPIUM HIRSUTUM L.) CALLUS PROTOPLASTS TO MACROCALLI". Thesis, The University of Arizona, 1985. http://hdl.handle.net/10150/275376.

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24

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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25

Yokoya, Kazutomo. "Studies on the regeneration of intergeneric somatic hybrid plants of roses". Thesis, University of East London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360915.

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26

Stanworth, Marie Helen. "Plasma membrane ATPase of Phytophthora cactorum". Thesis, University of the West of England, Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284886.

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27

Butler, Declan M. "Isolation and culture of protoplasts and tissues of Laminaria spp. (Phaeophyta)". Thesis, University of Leeds, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.237096.

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28

Sinha, Anupam. "Studies on the isolation and culture of Lupin (Lupinus albus) protoplasts". Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297700.

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29

Al-Forkan, Mohammad. "Agrobacterium-mediated transformation of Indica rice (Oryza sativa L.)". Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342480.

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30

Browne, H. M. "Protoplasts and their application to the study of genetics in Lipomyces starkeyi". Thesis, University of Nottingham, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235343.

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31

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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32

Matthews, Derek. "The use of protoplasts in the regeneration and genetic manipulation of rose". Thesis, University of East London, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385004.

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33

Jackson, R. T. "Isolation of plasma membranes from leaf protoplasts : A search for definitive markers". Thesis, University of Southampton, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373954.

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34

Slomka, Marek Jozef. "Studies of tomato golden mosaic virus in plants, protoplasts and tissue culture". Thesis, Imperial College London, 1987. http://hdl.handle.net/10044/1/46616.

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35

Edwards, K. J. "Studies of homologous recombination between plasmid and chromosomal DNA in plant protoplasts". Thesis, University of Leicester, 1990. http://hdl.handle.net/2381/35209.

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A vector, called pWreckl, was constructed for the direct DNA transformation of N. tabacum protoplasts. This vector was shown to confer kanamycin resistance to transformed callus. Using derivatives of this plasmid containing various reporter genes, a protocol for direct DNA transformation by PEG-mediated uptake was established. However, all attempts to obtain transformed callus by electroporation were unsuccessful. The CAB6 gene from N. tabacum was identified as a suitable candidate for gene targeting and was further manipulated to produce constructs in pWreckl which were used in gene targeting experiments. Using a derivative of pWreckl, called BinsupF, transformed tobacco plants were generated and used as a model system for the rescue of integrated sequences by supF selection. This system was found to be capable of rescuing integrated pWreckl sequences, but at an efficiency below that which would be required for the rescue of rare gene targeting events. Therefore a second protocol was developed which used the technique of polymerase chain amplification to amplify specific integration events. Following direct DNA transformation with a pWreckl.CAB6 construct, a number of possible gene targeting events were identified and subcloned into pUC13. Partial sequencing of these clones revealed that they were not the result of homologous recombination, but arose through a combination of plasmid degradation and rearrangement which appeared to precede the actual integration event.

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36

Collings, Alan. "Cellulase and related enzyme activity in protoplasts and mycelium of filamentous fungi". Thesis, Sheffield Hallam University, 1986. http://shura.shu.ac.uk/19494/.

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Production of cellulase, pectinase and xylanase was investigated using culture filtrates from selected mesophilic (Aspergillus niger, Penicillium funiculosum, Penicillium janthinellum, Penicillium ochrochloron, Trichoderma viride), thermotolerant (Aspergillus fumigatus) and thermophilic (Geosmithia emersonii, formerly Talaromyces emersonii; the perfect stage of Penicillium emersonii) fungi. The fungi were grown on Acetobacter xylinum cellulose pellicles and soluble carboxymethyl cellulose and their enzyme complements were characterised under various growth conditions. The factors affecting the production and release of protoplasts from the closely related A. fumigatus and A. niger and P. ochrochloron and G. emersonii were investigated. Protoplast yields were found to be greatly dependent upon growth temperature, cultural conditions, mycelial age, mycelial concentration, incubation time, incubation temperature and lytic enzyme used. Greatest protoplast yields were obtained after 3 h incubation using 1 mg ml-1 Novozym TM 234 in 0.1 M 2-N-[Morpholino] ethane sulphonic acid (MES) buffer, pH 5.0 containing 0.6 M sodium chloride. Supplementation of the incubation medium with 0.03 M calcium chloride enhanced protoplast release in P. ochrochloron but not in the other three test fungi. Protoplasts from all four fungi were capable of regenerating a cell wall in both liquid and solid regeneration media. Regenerating protoplasts were shown to retain endo-B-1,4-glucanase, exo-B-1,4-glucanase and B-glucosidase activity. Attempts were made to cross related mesophilic with thermotolerant and/or thermophilic fungi using the technique of protoplast fusion. P. ochrochloron was crossed with G. emersonii and A. niger was crossed with A. fumigatus in an attempt to improve cellulase complement and stability. The test fungi were screened for resistance to a variety of heavy metal salts and commercial fungicides, and mutants resistant to Benlate and Mystox TM fungicides were obtained. These selection markers, together with an auxotrophic mutant of P. ochrochloron, were used to select out protoplast fusion products against parental types in the trial crosses.

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37

Penmetsa, Ramachandra V. "Factors influencing transient gene expression in electroporated tall fescue (Festuca arundinacea Schreb.) protoplasts". Thesis, This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-09052009-040406/.

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38

Abram, Beverley Louise. "Responses of Nicotiana glauca guard cell protoplasts to changes in the culture environment". Thesis, Lancaster University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314238.

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39

Elshihy, O. M. "In vitro culture of tissues, cells and protoplasts of cotton (Gossypium barbadense L.)". Thesis, University of Southampton, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374060.

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40

Starling, Anthony Patrick. "The uptake of heavy metals by protoplasts and whole cells of filamentous fungi". Thesis, Keele University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293925.

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41

Valles, K. L. M. "The use lipophilic ions to measure intracellular membrane potentials in intact wheat protoplasts". Thesis, University of Sussex, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373919.

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42

Daniels, Alison. "Microscopic and computer analysis of ultrastructural changes accompanying isolation and manipulation of tobacco protoplasts". Thesis, University of Nottingham, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278755.

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43

Harker, C. L. "The detection of virus coded proteins in cauliflower mosaic virus infected plants and protoplasts". Thesis, Bucks New University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376417.

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44

Yeung, Elaine Yee-Ling. "Functional roles of group vii erfs through transient transformation of protoplasts in Arabidopsis and rice". Thesis, University of California, Riverside, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=1547838.

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Environmental stresses involving flooding and the accompanying cellular low oxygen (hypoxia) constitute a major limitation to agricultural production worldwide. These stresses hinder the plant's ability to maintain energy production and growth. Yet, previous studies have shown that certain genes of group VII ethylene responsive factor (ERF) family of transcription factors play a functional role in the survival of submergence and low oxygen stresses in Oryza sativa(rice) and Arabidopsis thaliana. This study utilized a transient gene transfection assay of protoplasts to characterize gene networks regulated by the group VII ERFs by measuring the transcriptional activation of various promoters. Experiments revealed that orthologous genes are regulated by the group VII ERFs of both species. In rice, an ERF-type transcription factor, SUB1A, confers submergence tolerance by regulating carbohydrate metabolism, elongation growth, and hormonal responses. SUB1A is linked to the related genes SUB1B and SUB1C. Here, two alleles of SUB1A (SUB1A-1 , SUB1A-2) as well as one allele each of the other two genes (SUB1B-1 and SUB1C-1) were shown to be transcriptional activators, and the nuclear localization of SUB1A-1 and SUB1C-1 was demonstrated. In Arabidopsis, core hypoxia responsive genes are governed by the N-end rule pathway of targeted proteolysis, in which proteins of the group VII ERFs (i.e., RAP2.12 and HRE2) are regulated post-translationally under hypoxic conditions. Transactivation experiments demonstrated that the trihelix protein HRA1 negatively regulates the transcriptional activation ability of RAP2.12 in the core hypoxia response. By contrast, HRE2 was not targeted by HRA1 downregulation, but was instead upregulated by RAP2.12 during the stress. Experiments involving truncations of HRA1 reveal that the coil-coil domain is functionally important for HRA1 interaction with group VII ERF complexes. These results with a rapid protoplast assay provide a foundation for future investigations into the low oxygen response gene network regulated by group VII ERF transcription factors binding to cis-regulatory elements, and modulated by the trihelix protein HRA1.

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45

Yan-Xiu, Zhao. "The isolation, culture and genetic manipulation of protoplasts from salt and drought tolerant leguminous plants". Thesis, Coventry University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332788.

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46

Shacklock, Philip S. "The involvement of second messengers in the phytochrome-mediated swelling of etiolated wheat leaf protoplasts". Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/14380.

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Although a considerable amount of circumstantial evidence exists which indicates the involvement of Ca²&43 in the regulation of phytochrome-mediated responses (Bossen et al., 1988; Viner et al., 1988; Roux, 1986), the exact nature of this involvement has remained unclear. Etiolated wheat leaf protoplasts swell in response to red light (Bossen et al., 1988) by a mechanism which is phytochrome-mediated and is related to leaf growth and unrolling (Zhou et al., 1990). In this study fluorescent Ca²+ indicators have been used in conjunction with Laser Scanning Confocal Microscopy to measure changes in [Ca²+ ]_i following red light irradiation of these protoplasts. In most cases (n = 18 of 23), an increase in [Ca²+ ]_i of approximately 400-700nM was observed, followed by a decrease to below resting level. This response preceded the physiological response of protoplast swelling, although the timing of the [Ca²+ ]_i response varied between protoplasts. Transient increases in [Ca²+ ]_i initiated by photoactivation of caged Ca²+ or caged InsP₃ mimicked the swelling response, and photoactivation of Diazo-2, a caged Ca²+ scavenger, within these protoplasts prevented the swelling response. Data is also presented here which suggests that cAMP, released from caged cAMP, can also induce an increase in protoplast volume, possibly by causing an increase in [Ca²+ ]_i. These data support the hypothesis that phytochrome signals are transduced through Ca²+ and suggest a possible role for cAMP in signal transduction within plant cells.

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47

Abdullah, R. B. "The development of a reproducible system for efficient plant regeneration from rice (Oryza sativa L.) protoplasts". Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381060.

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48

Woskow, Steven A. "Effect of Proteolytic Enzymes on Transfection and Transformation of Streptococcus lactis Protoplasts and Transformation of Streptococcus cremoris". DigitalCommons@USU, 1987. https://digitalcommons.usu.edu/etd/5357.

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The effect of proteolytic enzymes on the transformation and transfection of Streptococcus lactis LM2301 protoplasts was examined in an attempt to eliminate the variability observed. By using both chymotrypsin and mutanolysin to form protoplasts followed by spread plating, consistent frequencies of 104 to 105 transformants per μg of pGB301 DNA, and 105 transfectants per μg of c2 bacteriophage DNA where achieved. Optimum transformation and transfection frequencies were obtained when 16 h cultures were treated simultaneously with 25 U/ml mutanolysin and 1.25 U/ml chymotrypsin for 15 min. Trypsin and pronase in conjunction with mutanolysin also increased transformation frequencies higher than when mutanolysin was used alone, but pronase was not as effective as chymotrypsin or trypsin. These results may explain the variability in transformation of mutanolysin-treated cells of S. lactis since commercial sources of mutanolysin contain varying amounts of proteolytic enzyme activity. Transformation of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of pGB301 DNA) was achieved. Plasmid pGB301 was able to replicate and express antibiotic resistance in the resultant transformant (designated S. cremoris SW301). The presence of pGB301 in S. cremoris SW301 was confirmed by agarose gel electrophoresis.

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49

Gomez, Luengo Rodolfo Gustavo. "Proteins and serological relationships of maize mosaic virus isolates and replication of the virus in Maize (Zea Mays L.) protoplasts /". The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487327695621001.

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50

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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