Cultivos celulares meristemáticos de Peumus boldus Mol. obtenidos a partir de discos de capa fina procedentes de internodos y de genotipos distintos cultivados in vitro.
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Date
2024
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Universidad de Concepción
Abstract
Peumus boldus Mol. (boldo) es un árbol nativo que crece en el centro y sur de Chile, que durante años se ha utilizado con fines farmacológicos. La boldina es su principal alcaloide, ampliamente usado por su alta actividad antioxidante. Para su obtención se utiliza la recolección de hojas o corteza en bosques mixtos, lo que ha llevado a la sobreexplotación de la especie. Recientemente, se ha recurrido al cultivo de células meristemáticas del cámbium vascular (CMC) en plantas silvestres para la producción de metabolitos secundarios o especializados. Una alternativa para las plantas cultivadas in vitro es el cultivo de células meristemáticas del brote y de la raíz. Sin embargo, el aislamiento y cultivo de estas fuentes meristemáticas es complejo debido a que su presencia está limitada a una pequeña porción apical. Una posible solución es la utilización de discos de capa fina (Thin cell layer, en inglés), transversales y proximales al meristema apical para iniciar cultivos celulares. Debido a esto, el objetivo de esta investigación es obtener y caracterizar cultivos celulares de P. boldus obtenidos a partir de discos de capa fina desde internodos y genotipos distintos cultivados in vitro. Mediante 3 objetivos específicos: 1. Comparar la morfología y el crecimiento de cultivos celulares obtenidos de discos de capa fina de diferentes internodos de microplantas de tres genotipos de P. boldus. 2. Caracterizar cito-histológicamente las respuestas de los cultivos celulares obtenidos de discos de capa fina de diferentes internodos de microplantas de tres genotipos de P. boldus. 3. Contrastar a nivel histoquímico la respuesta a la elicitación de los cultivos celulares obtenidos de discos de capa fina de diferentes internodos de microplantas de tres genotipos de P. boldus.
Los principales resultados de la investigación establecieron que los discos de capa fina del primer internodo de genotipos específicos desarrollan una proliferación friable, óptima para iniciar cultivos celulares, de forma más rápida y con un tamaño incluso mayor que los discos iniciales. Seguidos por el segundo y, finalmente, por el tercer internodo, que requiere más tiempo para desarrollarse. Posteriormente, se realizaron cultivos celulares con los diferentes tipos de internodos y se observaron células globulares con tendencia a la aglomeración, así como células alargadas, ambas con acumulación de diversos metabolitos secundarios. Los resultados histoquímicos muestran una acumulación de alcaloides en todos los cultivos, no hubo presencia de fenoles ni proteínas, además que la acumulación de algunos metabolitos depende del tipo de proliferación celular del cultivo. Mientras que, en otros, el uso de metil jasmonato y tirosina podría influir en la competencia entre rutas metabólicas.
Las principales conclusiones son que la proliferación friable obtenida de los primeros internodos se debe a una mayor presencia de células indiferenciadas procedentes del cámbium fascicular e interfascicular, las cuales provienen de discos de capa fina de brotes de 4 cm de altura con un tamaño de médula pequeño en relación con la corteza del brote inicial. En relación con los cultivos celulares, se determinó que las células globulares corresponden a células meristemáticas, mientras que las células alargadas corresponden a células desdiferenciadas del córtex. Respecto a los análisis histoquímicos, la acumulación de alcaloides no se ve influenciada por ningún patrón hormonal evaluado, lo que sugiere que la producción se debe a las múltiples funciones de los alcaloides en respuesta al estrés. Además, la elicitación con metil jasmonato y tirosina tiende a causar una inhibición o estimulación en la síntesis de ciertos compuestos. Esta investigación constituyó un estudio exploratorio de pruebas histoquímicas en cultivos celulares meristemáticos.
Peumus boldus Mol. (boldo), a tree native to Chile, has been used for years for pharmacological purposes due to its main alkaloid, boldine, known for its high antioxidant activity. However, collecting leaves and bark in mixed forests has led to overexploitation of the species. Recently, the cultivation of vascular cambium meristematic cells (CMC) in wild plants has been explored as an alternative to produce secondary metabolites. Although the cultivation of shoot and root meristematic cells in plants grown in vitro is another option, their isolation and culture are complicated due to their presence limited to a small apical portion. A possible solution is the use of thin layer discs, extracted transversely and close to the apical meristem, to initiate cell cultures. Due to this, the objective of this research is to obtain and characterize cell cultures of P. boldus obtained from thin layer discs from internodes and different genotypes grown in vitro. Which was addressed through 3 specific objectives: 1. Compare the morphology and growth of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. 2. Cyto-histologically characterize the responses of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. 3. Contrast at a histochemical level the response to elicitation of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. The main results of the research established that the thin layer discs of the first internode of specific genotypes develop a friable proliferation, optimal for starting cell cultures, more quickly and with an even larger size than the initial discs. Followed by the second and finally the third internode, which requires more time to develop. Subsequently, cell cultures were carried out with the different types of internodes and globular cells with a tendency to agglomeration, as well as elongated cells, both with accumulation of various secondary metabolites, were observed. The histochemical results show an accumulation of alkaloids in all cultures, there was no presence of phenols or proteins, and the accumulation of some metabolites depends on the type of cell proliferation of the culture. While, in others, the use of methyl jasmonate and tyrosine could influence the competition between metabolic pathways. The main conclusions are that the friable proliferation obtained from the first internodes is due to a greater presence of undifferentiated cells from the fascicular and interfascicular cambium, which come from thin layer discs of buds 4 cm high with a small pith size in relation to the cortex of the initial shoot. In relation to the cell cultures, it was determined that the globular cells correspond to meristematic cells, while the elongated cells correspond to dedifferentiated cells of the cortex. Regarding histochemical analyses, alkaloid accumulation is not influenced by any hormonal pattern evaluated, suggesting that production is due to the multiple functions of alkaloids in response to stress. Furthermore, elicitation with methyl jasmonate and tyrosine tends to cause an inhibition or stimulation in the synthesis of certain compounds. This research constituted an exploratory study of histochemical tests in meristematic cell cultures.
Peumus boldus Mol. (boldo), a tree native to Chile, has been used for years for pharmacological purposes due to its main alkaloid, boldine, known for its high antioxidant activity. However, collecting leaves and bark in mixed forests has led to overexploitation of the species. Recently, the cultivation of vascular cambium meristematic cells (CMC) in wild plants has been explored as an alternative to produce secondary metabolites. Although the cultivation of shoot and root meristematic cells in plants grown in vitro is another option, their isolation and culture are complicated due to their presence limited to a small apical portion. A possible solution is the use of thin layer discs, extracted transversely and close to the apical meristem, to initiate cell cultures. Due to this, the objective of this research is to obtain and characterize cell cultures of P. boldus obtained from thin layer discs from internodes and different genotypes grown in vitro. Which was addressed through 3 specific objectives: 1. Compare the morphology and growth of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. 2. Cyto-histologically characterize the responses of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. 3. Contrast at a histochemical level the response to elicitation of cell cultures obtained from thin layer discs of different internodes of microplants of three genotypes of P. boldus. The main results of the research established that the thin layer discs of the first internode of specific genotypes develop a friable proliferation, optimal for starting cell cultures, more quickly and with an even larger size than the initial discs. Followed by the second and finally the third internode, which requires more time to develop. Subsequently, cell cultures were carried out with the different types of internodes and globular cells with a tendency to agglomeration, as well as elongated cells, both with accumulation of various secondary metabolites, were observed. The histochemical results show an accumulation of alkaloids in all cultures, there was no presence of phenols or proteins, and the accumulation of some metabolites depends on the type of cell proliferation of the culture. While, in others, the use of methyl jasmonate and tyrosine could influence the competition between metabolic pathways. The main conclusions are that the friable proliferation obtained from the first internodes is due to a greater presence of undifferentiated cells from the fascicular and interfascicular cambium, which come from thin layer discs of buds 4 cm high with a small pith size in relation to the cortex of the initial shoot. In relation to the cell cultures, it was determined that the globular cells correspond to meristematic cells, while the elongated cells correspond to dedifferentiated cells of the cortex. Regarding histochemical analyses, alkaloid accumulation is not influenced by any hormonal pattern evaluated, suggesting that production is due to the multiple functions of alkaloids in response to stress. Furthermore, elicitation with methyl jasmonate and tyrosine tends to cause an inhibition or stimulation in the synthesis of certain compounds. This research constituted an exploratory study of histochemical tests in meristematic cell cultures.
Description
Tesis presentada para optar grado de Magister en Ciencias Forestales
Keywords
Boldo, Biotecnología vegetal, Cultivo de tejidos vegetales