Efecto sobre la calidad fisiológica de semillas de soja (Glycine max L. Merr.) sometidas a un protocolo de extracción de ADN
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2019
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Abstract
La soja (Glycine max L. Merr.) es la principal oleaginosa cultivada en el mundo, con
un gran interés nutricional debido a su alto contenido de proteína y aceite, razón por la cual se
difundió ampliamente, siendo actualmente Estados Unidos, Brasil y Argentina los principales
productores a nivel mundial. Es una especie autógama y de escasa variabilidad genética
debido principalmente a la estrecha base de cultivares utilizados como parentales en los
programas de mejoramiento. Desde el comienzo del mejoramiento vegetal se utilizaron los
marcadores morfológicos para el proceso de selección (por ejemplo, el color de flor y la
forma de hoja), los cuales consisten en características fenotípicas de sencilla identificación
visual. Sin embargo, una limitante importante en el uso de estos marcadores fenotípicos radica
en su escasa disponibilidad, normalmente asociada a que se trata de genes de herencia
monogénica y predecible según las leyes de Mendel. Adicionalmente, muchas veces la
visualización del carácter se debe realizar en estadio de planta adulta, lo que prolonga los
tiempos de evaluación, aumenta la necesidad de mano de obra e incrementa
considerablemente el espacio necesario para conducir los programas de mejoramiento. Otro
aspecto de relevancia, es el efecto del ambiente, que puede dificultar la clara visualización y
confirmación de caracteres morfológicos, quedando muchos de éstos ligados a la subjetividad,
capacidad y entrenamiento del observador. A partir de la década del 80, el advenimiento de
las tecnologías de marcadores moleculares permitió agilizar y optimizar el progreso genético
obtenido a través de la selección. Este tipo de marcadores se detectan directamente a nivel del
ADN y puede corresponder a secuencias tanto codificantes como no codificantes. El gran
avance y desarrollo de estas tecnologías, sumado a la capacidad de análisis, exactitud,
reproducibilidad y costo accesible de los marcadores moleculares, derivó en su rápida
adopción e implementación en el mejoramiento vegetal en general e incluso para la
verificación e identificación de variedades de soja. Por otra parte, etapas cruciales en el
mejoramiento de soja requieren de la evaluación sistematizada de individuos únicos con el
objetivo de identificar aquellos que continuarán en el proceso de selección y mejoramiento.
Esto implica el minucioso cuidado de los mismos durante todo su ciclo de vida, a fin de
mantener la estricta trazabilidad genotípica de los individuos de interés, la cual es
normalmente evaluada a partir del tejido foliar. En este sentido, la posibilidad de realizar
análisis moleculares a partir del propio tejido seminal optimizaría el proceso de mejoramiento,
permitiendo no sólo un mejor seguimiento de los individuos, sino, además, aumentar la
eficiencia del proceso en general al identificar y seleccionar tempranamente aquellos
individuos de real interés. Esto requiere de manera excluyente, garantizar la calidad de cada
uno de estos individuos únicos e irreemplazables que aseguren su posterior siembra y
desarrollo de manera exitosa. Por esta razón, el objetivo del presente trabajo fue evaluar el
efecto sobre la calidad fisiológica de semillas de soja sometidas a un protocolo de extracción
de ADN para análisis moleculares basados en PCR.
El tratamiento de las semillas consistió en la realización de una perforación de un
milímetro de diámetro en la zona de los cotiledones lejana a la zona de inserción con el eje
embrionario. Semillas del mismo cultivar que no fueron sometidas al tratamiento fueron
utilizadas como control. El tejido extraído de cada semilla se sometió a un protocolo de
extracción de ADN para determinar si es apto para ser utilizado en técnicas de marcadores
moleculares basadas en PCR. La calidad fisiológica de las semillas se evaluó al inicio del
tratamiento y a los 30, 60, 90 y 180 días después del tratamiento, a través de las pruebas de
Poder Germinativo y Energía Germinativa, prueba de Vigor por Envejecimiento Acelerado,
prueba de Conductibilidad Eléctrica y Prueba de Viabilidad y Vigor por Tetrazolio.
La cantidad y calidad de ADN extraído resultaron adecuadas para su utilización en
análisis moleculares basados en PCR. Los análisis de calidad fisiológica mostraron que el
tratamiento de extracción de tejido no afectó notoriamente la calidad de las semillas, con
valores similares de viabilidad y germinación y un deterioro progresivo levemente mayor en
el tiempo para las semillas tratadas respecto al control. Estos resultados avalarían, por lo
tanto, la implementación de un protocolo de extracción de ADN para análisis moleculares
basados en PCR a partir de extractos de tejidos de semillas sin afectar la calidad de las
mismas para su uso como simiente.
Soybean (Glycine max L. Merr.) is the most important oilseed plant in the world, having a high nutritional value due to its protein and oil content, which has determined its wide adoption worldwide, with the United States, Brazil and Argentina being the most important producers. It is a self-pollinated species with low genetic variability, as a consequence of the narrow genetic bases used in most breeding programs. Morphological markers have been used since the dawn of plant breeding (for example, the color of flowers or the shape of leaves), which consist of phenotypic characteristics that are easily detected by visual inspection. Nonetheless, an important limitation in the application of these types of markers is their low availability, usually associated with the fact that they come from genes of monogenic inheritance as described by the laws of Mendel. Additionally, many times the visualization of the phenotype has to be done at the adult stage of the plant, which extends the evaluation times, increases the labor needs and requires larger spaces to conduct a breeding program. Another relevant fact is the effect of the environment on these types of markers, which can interfere with their visualization, giving room to mistakes, depending on the subjectivity and skill of the breeder. Since the '80s, the development and implementation of molecular markers allowed breeders to increase the genetic progress obtained through selection. These types of markers are detected at the DNA level and may correspond to coding or non-coding sequences. The rapid development of these technologies, together with their accuracy, high reproducibility and affordability, determined their current high adoption in breeding programs as wells as their use in varietal identification. On the other hand, some of the crucial steps in soybean breeding require the systematic evaluation of unique individuals, with the objective of identifying those who will continue in the breeding and selection process. This implies a careful manipulation of individuals throughout their lifecycle, to guaranty the genotypic traceability, which is normally evaluated from leaf tissue. Therefore, the availability of a method to use molecular markers directly from seed tissue could optimize the breeding processes, allowing improving the follow-up of individuals, increasing the efficiency in general by early identification and selection of high-value individuals. This requires the need to guarantee the survival and adequate development of individual seeds subject to analysis. To this end, the objective of the current work was to determine the effect on the physiological quality of seeds subjected to a tissue sampling protocol that is both adequate for DNA extraction and molecular analysis. Treated seeds were subjected to a 1-millimeter perforation on the cotyledon, away from the embryonic axis insertion point. Untreated seeds of the same cultivar were used as controls. The tissue sampled was used for DNA extraction in order to determine its suitability for PCR based molecular marker analysis. The physiological quality of the seeds was evaluated at 0, 30, 60, 90 and 180 days post-treatment, through the following tests: Germination Test, Germination Energy, Accelerated Ageing, Electrical Conductivity and Tetrazolium Test. We determined that the quantity and quality of the DNA extracted was adequate for PCR based molecular marker analysis. The physiological quality analysis showed that the tissue sampling did not affect significantly the quality of the seeds, with similar values of viability and germination between treated and control seeds, and a slightly higher deterioration with time for treated seeds. These results, therefore, endorse the implementation of a seed sampling and DNA extraction protocol that does not affect significantly the quality of seeds.
Soybean (Glycine max L. Merr.) is the most important oilseed plant in the world, having a high nutritional value due to its protein and oil content, which has determined its wide adoption worldwide, with the United States, Brazil and Argentina being the most important producers. It is a self-pollinated species with low genetic variability, as a consequence of the narrow genetic bases used in most breeding programs. Morphological markers have been used since the dawn of plant breeding (for example, the color of flowers or the shape of leaves), which consist of phenotypic characteristics that are easily detected by visual inspection. Nonetheless, an important limitation in the application of these types of markers is their low availability, usually associated with the fact that they come from genes of monogenic inheritance as described by the laws of Mendel. Additionally, many times the visualization of the phenotype has to be done at the adult stage of the plant, which extends the evaluation times, increases the labor needs and requires larger spaces to conduct a breeding program. Another relevant fact is the effect of the environment on these types of markers, which can interfere with their visualization, giving room to mistakes, depending on the subjectivity and skill of the breeder. Since the '80s, the development and implementation of molecular markers allowed breeders to increase the genetic progress obtained through selection. These types of markers are detected at the DNA level and may correspond to coding or non-coding sequences. The rapid development of these technologies, together with their accuracy, high reproducibility and affordability, determined their current high adoption in breeding programs as wells as their use in varietal identification. On the other hand, some of the crucial steps in soybean breeding require the systematic evaluation of unique individuals, with the objective of identifying those who will continue in the breeding and selection process. This implies a careful manipulation of individuals throughout their lifecycle, to guaranty the genotypic traceability, which is normally evaluated from leaf tissue. Therefore, the availability of a method to use molecular markers directly from seed tissue could optimize the breeding processes, allowing improving the follow-up of individuals, increasing the efficiency in general by early identification and selection of high-value individuals. This requires the need to guarantee the survival and adequate development of individual seeds subject to analysis. To this end, the objective of the current work was to determine the effect on the physiological quality of seeds subjected to a tissue sampling protocol that is both adequate for DNA extraction and molecular analysis. Treated seeds were subjected to a 1-millimeter perforation on the cotyledon, away from the embryonic axis insertion point. Untreated seeds of the same cultivar were used as controls. The tissue sampled was used for DNA extraction in order to determine its suitability for PCR based molecular marker analysis. The physiological quality of the seeds was evaluated at 0, 30, 60, 90 and 180 days post-treatment, through the following tests: Germination Test, Germination Energy, Accelerated Ageing, Electrical Conductivity and Tetrazolium Test. We determined that the quantity and quality of the DNA extracted was adequate for PCR based molecular marker analysis. The physiological quality analysis showed that the tissue sampling did not affect significantly the quality of the seeds, with similar values of viability and germination between treated and control seeds, and a slightly higher deterioration with time for treated seeds. These results, therefore, endorse the implementation of a seed sampling and DNA extraction protocol that does not affect significantly the quality of seeds.
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Keywords
Soja, Tejido seminal, Calidad fisiológica, Extracción de ADN, Fitomejoramiento, Análisis