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.

No description available.

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. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-02-12T12:22:13Z No. of bitstreams: 1 Dissertacao Barbara Lizandra Perini de Souza.pdf: 2248330 bytes, checksum: 2a20339f50d031a74d2a889ddbe2435e (MD5) / Made available in DSpace on 2015-02-12T12:22:13Z (GMT). No. of bitstreams: 1 Dissertacao Barbara Lizandra Perini de Souza.pdf: 2248330 bytes, checksum: 2a20339f50d031a74d2a889ddbe2435e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.

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. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.

Protoplasts were isolated from tobacco and barley leaves in sucrose or mannitol using commercially available cellulases and macerozymes. Barley growth and protoplast isolation and purification conditions were optimized so that protoplasts were obtained in high yields free of unwanted debris and organelles. A technique for processing barley and tobacco protoplasts for examination by scanning electron microscopy was developed in which protoplasts seem to have maintained their structural integrity. Barley and tobacco protoplasts took up 3H-B. subtilis DNA, 125I-B. subtilis DNA or 125I-M. luteus DNA as a linear function of time (0-6 hr) and DNA concentration (0-200 μg/ml). Up to 16 pg of exogenous DNA was taken up per protoplast of which approximately one half became nuclear associated. Protoplasts were viable after the uptake as shown by standard staining and culturing techniques. Approximately 20% of the DNA taken up after typical 4 hr uptake reactions was of average gene size (5-10 x 105 daltons), and therefore of potential significance to host gene expression.

Freshly isolated protoplasts from pea leaves were used to investigate the responses of antioxidant enzymes to oxidative stress. Two cultivars, Progress (tolerant) and Nugget (sensitive), that have differing resistance with respect to oxidative stress at the whole plant level were used. Sulfite and the superoxide generating herbicide, paraquat, were used as the oxidants. Final sulfite concentrations during photosynthetic incubations ranged from 1.5 mM to 30.0 mM. During the polarographic estimation of photosynthesis, CO₂-dependent O₂ evolution did not decrease. At sulfite concentrations of 3.0 mM or less, light-dependent O₂ evolution increased and was probably due to a concomitant SO₂-dependent O₂ evolution. Photosynthesis determined as ¹⁴CO₂ fixation was not increased at these low concentrations of sulfite. Concentrations greater than 7 mM = sulfite inhibited photosynthetic ¹⁴CO₂ fixation. No difference in these responses was found between the two cultivars. At 0.1 µM paraquat, the relative resistance to oxidative stress was reversed compared to previous studies at the whole plant level. With the tolerant cultivar, activity of the plastid antioxidant enzyme, glutathione reductase, increased after a three-hour exposure. Changes in the steady state level of glutathione reductase protein, as judged by immunoblots, did not correlate with the observed changes in enzyme activity. No change in the de novo synthesis of glutathione reductase occurred over the same period as a consequence of paraquat application. A mechanism, unrelated to oxygen free radical scavenging, may contribute to the relative tolerance to low concentrations of paraquat. On the other hand, after an eight-hour exposure to 0.1 mM PQ in the presence of Gamborg’s basal salts, superoxide dismutase activity of Progress protoplasts was enhanced 288% above the preexposure levels while glutathione reductase activity decreased 70% and ascorbate peroxidase activity decreased 90%. The relationship of these changes to oxidative damage to the photosynthetic machinery remains to be assessed. / Ph. D.

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase (GUS) gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 x 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3 and 5 day old suspensions. These results show that transient expression studies can be used to optimize electroporation parameters rapidly. Results of such rapid assays can be used as a basis for further studies on stable transformation of this important turf-grass species. / Master of Science

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.

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.

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.