Aislamiento y cultivo de protoplastos de Vanilla odorata y V. pompona
Fecha
2024
Tipo
tesis doctoral
Autores
Warner Pineda, Jorge Enrique
Título de la revista
ISSN de la revista
Título del volumen
Editor
Resumen
El objetivo de esta investigación fue producir un protocolo eficiente para el aislamiento de protoplastos de Vanilla odorata y de V. pompona y cultivarlos con el propósito de promover su uso en el mejoramiento genético de la vainilla. Se desarrolló por primera vez un protocolo para el aislamiento de protoplastos de V. odorata y se optimizó parámetros claves para el aislamiento de ambas especies con rendimientos de cerca de 300,000 protoplastos g 1 de tejido fresco y 85 % de viabilidad. Se caracterizó químicamente los polisacáridos de la pared celular de cuatro especies de Vanilla y se encontraron perfiles específicos de azúcares neutros y de ácidos urónicos que podrían tener un significado práctico para optimizar la digestión de la pared celular y para formular composiciones de medios adecuados para la regeneración de la pared celular durante el cultivo de protoplastos. Se desarrolló por primera vez protocolos para la regeneración directa e indirecta de V. odorata y la regeneración indirecta de V. pompona. Se evaluó la transferibilidad de 14 loci de microsatélites, originalmente desarrollados para V. planifolia, a 12 de las 13 especies de Vanilla nativas de Costa Rica y a un híbrido. La transferibilidad de estos marcadores reveló relaciones congruentes entre las especies y permitió la identificación de algunas especies. Se optimizó un protocolo de citometría de flujo que previene eficientemente el agrupamiento de núcleos y obstrucciones del instrumento causadas por mucílagos y la presencia de rafidios en el material vegetal de Vanilla. Con este método se midió el contenido de ADN-C del mismo grupo de 12 especies de Vanilla nativas de Costa Rica, el cual varió entre 5.2 y 9.0 pg permitiendo identificar algunas especies. Se comunica por primera vez el contenido de ADN-2C de seis especies y de un híbrido comercial. Se confirmó la ocurrencia del fenómeno de endorreplicación parcial en todas las muestras analizadas. Los resultados de esta investigación contribuyen al estudio del aislamiento y el cultivo de protoplastos de V. odorata y V. pompona, así como a la comprensión de las especies de Vanilla silvestres de Costa Rica menos estudiadas, algunas de las cuales presentan características útiles para el mejoramiento o la diversificación del cultivo de la vainilla.
The objective of this research was to produce an efficient protocol for the isolation of protoplasts from Vanilla odorata and V. pompona, and to cultivate them with the aim of promoting their use in vanilla genetic improvement. A protocol for the isolation of V. odorata protoplasts was developed for the first time, and key parameters for isolation of both species were optimized with yields of approximately 300,000 protoplasts per gram of fresh tissue and 85 % viability. The cell wall polysaccharides of four Vanilla species were chemically characterized, revealing specific profiles of neutral sugars and uronic acids that could have practical significance for optimizing cell wall digestion and formulating suitable medium compositions for cell wall regeneration during protoplast culture. Protocols for direct and indirect regeneration of V. odorata and indirect regeneration of V. pompona were developed for the first time. The transferability of 14 microsatellite loci, originally developed for V. planifolia, was evaluated across 12 out of 13 native Vanilla species from Costa Rica and one hybrid. Transferability of these markers revealed congruent relationships among species and allowed for species identification. A flow cytometry protocol was optimized to efficiently prevent nucleus clumping and instrument clogging caused by mucilage and raphides in Vanilla plant material. DNA content (2C) of the same group of 12 native Vanilla species from Costa Rica, varied between 5.2 and 9.0 pg, enabling species identification. DNA content of six species and one commercial hybrid was reported for the first time. The occurrence of partial endoreplication was confirmed in all analyzed samples. The results of this research contribute to the study of isolation and culture of protoplasts from V. odorata and V. pompona, as well as the understanding of lesser-studied wild Vanilla species from Costa Rica, some of which exhibit useful characteristics for vanilla cultivation improvement or diversification.
The objective of this research was to produce an efficient protocol for the isolation of protoplasts from Vanilla odorata and V. pompona, and to cultivate them with the aim of promoting their use in vanilla genetic improvement. A protocol for the isolation of V. odorata protoplasts was developed for the first time, and key parameters for isolation of both species were optimized with yields of approximately 300,000 protoplasts per gram of fresh tissue and 85 % viability. The cell wall polysaccharides of four Vanilla species were chemically characterized, revealing specific profiles of neutral sugars and uronic acids that could have practical significance for optimizing cell wall digestion and formulating suitable medium compositions for cell wall regeneration during protoplast culture. Protocols for direct and indirect regeneration of V. odorata and indirect regeneration of V. pompona were developed for the first time. The transferability of 14 microsatellite loci, originally developed for V. planifolia, was evaluated across 12 out of 13 native Vanilla species from Costa Rica and one hybrid. Transferability of these markers revealed congruent relationships among species and allowed for species identification. A flow cytometry protocol was optimized to efficiently prevent nucleus clumping and instrument clogging caused by mucilage and raphides in Vanilla plant material. DNA content (2C) of the same group of 12 native Vanilla species from Costa Rica, varied between 5.2 and 9.0 pg, enabling species identification. DNA content of six species and one commercial hybrid was reported for the first time. The occurrence of partial endoreplication was confirmed in all analyzed samples. The results of this research contribute to the study of isolation and culture of protoplasts from V. odorata and V. pompona, as well as the understanding of lesser-studied wild Vanilla species from Costa Rica, some of which exhibit useful characteristics for vanilla cultivation improvement or diversification.
Descripción
Palabras clave
PLANTAS, PLANTS, VANILLA, VAINILLA, GENOME, MOLECULAR MARKERS