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Culturas embriogênicas podem ser cultivadas por anos, mas acumulam características deletérias ao longo do tempo: crescimento reduzido; aumento da necrose; estruturas aberrantes; maturação impedida; e, eventualmente, perda completa do potencial embriogênico. Esse fenômeno ocorreu em uma linha de tecido de pupunha (Bactris gasipaes Kunth) após oito anos de cultivo. Esta tese investigou esta ocorrência através de três diferentes abordagens: (i) o uso de 5-azacitidina para reverter a hipermetilação relacionada à idade e análise da abundância de proteína em culturas de dois anos e oito anos para determinar quais processos de plantas foram os mais afetados pelo envelhecimento; (ii) otimizar a conversão de plântulas de embriões somáticos com vasos selados ou ventilados, a fim de fornecer uma fonte de plântulas para a criação de novas linhas de cultura e avaliar o efeito do ambiente de cultura sobre bioquímica celular; e (iii) prevenir o envelhecimento através do desenvolvimento de novas estratégias para otimizar a vitrificação de embriões somáticos. A 5-azacitidina estimulou o crescimento de embriões somáticos em culturas de oito anos de idade. No entanto, o crescimento de culturas embriogênicas de dois anos de idade foi interrompido. A análise proteômica mostrou que todos os tecidos foram submetidos à hipóxia, sugerindo que todos os tecidos dependiam da fermentação como meio de geração de energia. A fermentação leva à acidificação citosólica, que pode causar danos a importantes processos celulares, como o metabolismo. A análise por HPLC-ELSD mostrou que as culturas de dois anos tinham quantidades detectáveis de ribose, arabinose e sacarose, mas nenhum carboidrato foi detectado em culturas de oito anos de idade. Além disso, muitos genes conservados relacionados ao controle epigenético foram acumulados em culturas de dois anos em comparação com culturas de oito anos de idade, sugerindo que as culturas mais jovens mantiveram uma maior quantidade de controle epigenético. No segundo capítulo, os vasos ventilados levaram a um menor crescimento de massa fresca comparado aos vasos selados de cultura, mas apresentaram maior massa seca e maior emergência de clorofila, sugerindo uma maior taxa de conversão embrionária. Além disso, maior abundância do total de poliaminas, e especialmente de putrescina e menor quantidade de frutose disponíveis e glicose sugerem que as culturas em vasos ventilados foram submetidos a grandes taxas de mudança, mas suas fontes endógenas de nutrientes pode ser insuficiente para abastecer a conversão completa de um embrião para um plantlet. No terceiro capítulo, várias estratégias de otimização mostraram grande potencial para melhorar o protocolo de vitrificação da pupunha. Culturas vitrificadas descongeladas na temperatura mais quente (75 ° C) tiveram as maiores taxas de rebrotação. Uma solução de vitrificação planta três diluída a 80% da sua concentração, incluindo 0,1 M de KCl, MgCl2, MgSO4 ou K2H (PO4) melhorou recrescimento. Além disso, a desidratação parcial melhorou o recrescimento das culturas possivelmente porque foi menos traumático para o tecido em comparação com a exposição com alta concentração de solutos. Os resultados desses três capítulos oferecem uma explicação do motivo pelo qual as culturas embriogênicas envelhecem e várias estratégias distintas para evitá-las.<br> |
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Abstract : The ability to grow plants in vitro is a powerful technology: rare, economically-important, or endangered plants may be propagated for conservation or reforestation; valuable genotypes may be mass-produced for agriculture; and models for studying various aspects of plant genetics, biochemistry, and morphology can be maintained indefinitely?or nearly. Cultures may be grown for years, but most begin to show their age; they begin to grow slower; their structures become malformed; and they begin to lose the ability to grow specialized structures. In this report, I explore the concept of impermanence in tissue culture using the model species peach palm (Bactris gasipaes), with a focus on studying and exploring means around impermanence. This doctorate dissertation is divided into four sections: (i) a bibliographic review; (ii) a study on the use of 5-azacytidine to reverse age-related loss of embryogenic potential and a comparison of differentially-accumulated proteins between an eight-year-old and a two-year-old culture line; (iii) a study comparing the growth response and differential biochemistry of mature peach palm cultures in ventilated and sealed vessels; and (iv) a study of several little-studied or novel factors to improve cryopreservation of embryogenic peach palm cultures. Peach palm cultures treated with 5-azacytidine, a potent inhibitor of cytosine methylation, showed several key results: two-year-old embryogenic tissue tended to lose morphological regulation with increasing concentrations, leading to the growth of non-embryogenic tissue and fast-growing yellow callus; overall increased tissue necrosis in two-year-old embryogenic tissue; and the growth of somatic embryos in 8.80% of eight-year-old-cultures treated with 16 µM 5-azacytidine?the first growth of somatic embryos in this culture line in two years. Two-year-old embryogenic cultures, old cultures, and the embryos derived from 5-azacytidine-treated old cultures were compared using shotgun proteomics. All three cultures showed signs that they were growing under hypoxic conditions by the presence of pyruvate decarboxylase and alcohol dehydrogenase, but either embryogenic tissue was better equipped for these conditions through up-accumulated proteins involved in maintaining the cellular redox state and epigenetic regulation. Old cultures showed a further down-accumulation of proteins involved in many aspects of metabolism, and an HPLC carbohydrate showed no detectable amounts of any of any carbohydrate in eight-year-old cultures, though young embryogenic and non-embryogenic cultures alike showed detectable amounts of ribose, arabinose, and sucrose. Further, many cell wall-related proteins were up-accumulated in eight-year-old tissue, possibly hinting at a connection between the tissue?s fibrous texture and its cellular state. These findings show that, over time, continuous hypoxic conditions create an unhealthy environment where a cell must conduct cytosol-acidifying cellular reactions to produce energy, and this decreased energy production may lead to a loss in ability for the cell to maintain its redox state, which may exacerbate or be exacerbated by a breakdown in epigenetic control, leading to cell behavior disruption and a loss of embryogenic potential. The second chapter details an analysis of the effects of a ventilated or closed environment in stimulating somatic embryo conversion to plantlets. Somatic embryos placed in ventilated vessels had overall less fresh mass gain, but those same cultures had overall higher dry weight, increased number of developing photosynthetic embryos, and contained overall higher concentrations of polyamines. In addition, though it was at the border of statistical significance, embryos placed in ventilation may have expended some of their energy reserves, as suggested by a decreased mean reduction in glucose (p=0.0624) and fructose (p=0.0587). Global methylation between either tissue was not significant, but both cultures in sealed or ventilated vessels had higher global methylation rates compared to cultures undergoing multiplication. The third chapter contains a series of experiments aimed at improving previously-established cryopreservation protocols through two general perspectives: the physics of water phase transitions and the biological aspect of cellular integrity. Speed is of utmost importance in both freezing and thawing: the faster a sample was thawed, the more likely that it?d regrow, and, consequently, the faster it was frozen, the more likely it would regrow as suggested by the decreased regrowth when droplet vitrification aluminum strips were replaced with polystyrene or less-conductive stainless steel. Embryogenic cluster size likewise had a significant impact on regrowth: though smaller sized clusters seemed to absorb vitrification solution faster, both vitrified and non-vitrified cultures alike faced severe drops in regrowth as incubation duration increased. Large cultures, however, had high regrowth rates at all durations. I also made two general attempts at improving plant vitrification solution 3 (PVS3; 50% w/v glycerol, 50% w/v sucrose)?a required, though toxic solution required for peach palm culture vitrification cryopreservation?using two general approaches: the use of dissolved salts in a reduced-concentration form of PVS3, and the use of heavy water and magnesium chloride. Compared to PVS3, 80% PVS3 showed initially lower regrowth rates, but this climbed as incubation duration increased, though regrowth potential in either were less than any of the 80%PVS3 mixtures with 0.1 M KCl, MgCl2, MgSO4, or K2H(PO4). This may be due to the use of various charged particles in maintaining macromolecule conformation, maintenance of electrical charge balance between the cell and the solution to prevent electrostatic-driven loss of dissolved ions; or maintenance of the charged phospholipid bilayer during rapid changed in a high-osmotic environment, but, simply put, there?s no way to know until a more in-depth experiment is conducted. The result of the heavy water experiment was less pronounced: MgCl2had no significant effect, and heavy water, though having a significant positive effect when MgCl2 was ignored as a factor, was not large enough to justify its costly use as part of a protocol. Dehydration pre-treatments for one or two hr improved regrowth rates for cultures incubated for 60 or 120 min. in vitrification solution, but longer dehydrations and longer incubation periods showed a large loss in regrowth. Based on these results, an improved cryopreservation protocol includes the following: faster thaw rates; vitrification strips made from thermally-conductive materials; little tissue manipulation; reduced 80%PVS3 concentration with 0.1 M of any of the four tested dissolved salts; and partial dehydration. |
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