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Universidad Autónoma Del Estado De México Maestría Y Caracterización molecular y cultivo in vitro de Yoloxóchitl ( Magnolia mexicana DC.) UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MÉXICO MAESTRÍA Y DOCTORADO EN CIENCIAS AGROPECUARIAS Y RECURSOS NATURALES CARACTERIZACIÓN MOLECULAR Y CULTIVO in vitro DE YOLOXÓCHITL (Magnolia mexicana DC.) TESIS QUE PARA OBTENER EL GRADO DE MAESTRO EN CIENCIAS ARGROPECUARIAS Y RECURSOS NATURALES PRESENTA: LILIA DANAE ARTEAGA RIOS COMITÉ DE TUTORES: Dr. Jaime Mejía Carranza. Tutor académico. Dr. José Luis Piña Escutia. Tutor adjunto. Dr. Justino Gerardo González Díaz. Tutor adjunto. Centro Universitario Tenancingo, Estado de México. Mayo 2019 i Caracterización molecular y cultivo in vitro de Yoloxóchitl ( Magnolia mexicana DC.) RESÚMEN La Magnolia mexicana , es una especie de importancia ecológica, medicinal e histórica, amenazada de extinción de acuerdo con la NOM-059-SEMARNAT-2010, debido a la pérdida de su hábitat, la tala clandestina y la dificultad reportada para propagarla por semilla; sumado a esto, la reciente reclasificación a nuevas especies a partir de lo que se consideraba una sola, ha significado la reducción de su área de distribución a Veracruz, Oaxaca y Puebla, con interrogantes a los límites de su distribución debido a la presencia de ejemplares posiblemente de la misma especie en el Estado de México, que tendrían relación además por sus antecedentes históricos. Así, a fin de ayudar a dar claridad sobre los alcances de su ámbito de ocurrencia, y contribuir a la resolución de sus problemas de propagación, este estudio tuvo como objetivos: 1) establecer las relaciones moleculares existentes entre dos individuos de Magnolia sp. presentes en Malinalco y Zumpahuacán, Estado de México, con individuos de poblaciones de M. mexicana en Zongolica, Veracruz con el empleo de RAPDs; y 2) probar diferentes explantes y medios de cultivo para la propagación in vitro de la especie. En el análisis molecular se utilizó el método CTAB para la extracción de ADN, que se amplificó mediante PCR con 14 cebadores RAPD; las relaciones moleculares entre las muestras se evaluaron estadísticamente con el método de agrupación Neighbour Joining, índice de similitud Dice y remuestreo de 1,000 corridas, esta metodología permitió discriminar entre las muestras de Magnolia spp. analizadas, con un PIC promedio de 0.30 que lo catalogaría como altamente informativo, con los cebadores OPA7, OPA8 y E18 con la mayor capacidad discriminante; las evidencias moleculares, contrastadas con datos morfológicos permitieron determinar que los ejemplares de Yoloxóchitl de Veracruz, Zumpahuacán y Malinalco, pertenecen al género Magnolia sección Talauma ; pero no fueron suficientes para asegurar que se tratara de la misma especie. En cuanto al cultivo in vitro se probaron siete diferentes métodos de desinfección en yemas axilares, se evaluó la efectividad de agentes inhibidores de fenoles (AIF), y la respuesta de yemas axilares a cuatro diferentes medios de cultivo, y se analizó la respuesta de distintos tejidos (vegetativos, florales y seminales) a diferentes combinaciones hormonales. Así, se encontró que el tratamiento con NaClO 20% (v/v), plata ionizada y 5 g/l de Terbinafina logró controlar la contaminación del 76% de explantes (p ˂ 0.05) ; sin embargo, no se logró determinar un AIF adecuado para tratar los explantes de M. mexicana en cultivo in vitro . También, se observó que una concentración de 0.5 mg/l de bencilaminopurina (BAP) en medio Murashigue-Skoog (MS) fue suficiente para la formación de brotes a partir de yemas axilares de Yoloxóchitl y la presencia de ádico naftalén acético (ANA) en el medio de cultivo a una concentración de 0.25 g/l promovió la formación de callo en este mismo tipo de explante; de la misma forma, la auxina sintética 2.4-D a una concentración de 3 mg/l adicionada con 1 mg/l de BAP, mostró ser útil en la inducción de callo en distintos tipos de explantes. Con este estudio, se dan avances hacia la creación de un protocolo para la micropropagación de M. mexicana , y queda abierta la posibilidad de que las magnolias de Malinalco y Zumpahuacán sean especies diferentes a M. mexicana. iv Caracterización molecular y cultivo in vitro de Yoloxóchitl ( Magnolia mexicana DC.) ABSTRACT The Magnolia mexicana is a species of ecological, medicinal and historical importance, threatened with extinction in accordance with NOM-059-SEMARNAT-2010, due to the loss of its habitat, clandestine logging and the reported difficulty in propagating it by seed; added to this, the recent reclassification to new species from what was considered a single, has involved the reduction of its area of distribution to Veracruz, Oaxaca and Puebla, with questions to the limits of its distribution due to the presence of specimens possibly of the same species in the State of Mexico, which would also be related to their historical background. Thus, in order to help to clarify the scope of its occurrence, and contribute to the resolution of its propagation problems, this study had as objectives: 1) to establish the molecular relationships between two individuals of Magnolia sp. present in Malinalco and Zumpahuacán, State of Mexico, with individuals from populations of M. mexicana in Zongolica, Veracruz with the use of RAPDs; and 2) test different explants and culture media for the in vitro propagation of the species. In the molecular analysis the CTAB method was used for DNA extraction, which was amplified by PCR with 14 RAPD primers; the molecular relationships between the samples were statistically evaluated with the Neighbor Joining method, similarity index Dice and resampling of 1,000 runs, this methodology allowed to discriminate between the samples of Magnolia spp. analyzed, with an average PIC of 0.30 that would classify it as highly informative, with the primers OPA7, OPA8 and E18 with the highest discriminant capacity; the molecular evidences, contrasted with morphological data allowed to determine that the Yoloxóchitl of Veracruz, Zumpahuacán and Malinalco, belong to the genus Magnolia, Talauma section; but they were not enough to ensure that it was the same species. Regarding in vitro culture, seven different disinfection methods were tested in axillary buds, the effectiveness of phenol inhibitors (AIF) was evaluated, and the response of axillary buds to four different culture media, and the response of different tissues (vegetative, floral and seminal) to different hormonal combinations. Thus, it was found that the treatment with NaClO 20% (v/v), ionized silver 3.5 ml/l and 5 g/l of Terbinafina managed to control the contamination of 76% of explants (p ˂ 0.05); however, it was not possible to determine an adequate AIF to treat explants of M. mexicana in in vitro culture. Also, it was observed that a concentration of 0.5 mg/l of bencilaminopurine (BAP) in Murashigue-Skoog medium (MS) was sufficient for the formation of shoots from Yoloxóchitl axillary buds and the presence of naftalen acetic adic (NAA) in the culture medium at a concentration of 0.25 g/l promoted callus formation in this same type of explant; in the same way, the synthetic auxin 2.4-D at a concentration of 3 mg/l added with 1mg/l of BAP, proved to be useful in the induction of callus in different types of explants. With this study, advances are made towards the creation of a protocol for the micropropagation of M. mexicana , and the possibility remains that the magnolias of Malinalco and Zumpahuacán are different species from M. mexicana . v Caracterización molecular y cultivo in vitro de Yoloxóchitl ( Magnolia mexicana DC.) CONTENIDO DEDICATORIA ................................................................................................................. I AGRADECIMIENTOS ..................................................................................................... II RESÚMEN ...................................................................................................................... IV ABSTRACT ..................................................................................................................... V ÍNDICE DE FIGURAS .................................................................................................... IX ÍNDICE DE CUADROS ................................................................................................. XII ÍNDICE DE ANEXOS ................................................................................................... XIII 1. INTRODUCCIÓN. ........................................................................................................ 1 2. REVISIÓN DE LITERATURA. ..................................................................................... 3 2.1. EL YOLOXÓCHITL . .................................................................................................... 3 2.1.1. Descripción de la especie. .............................................................................. 4 2.1.2. Distribución. .................................................................................................... 9 2.1.3. Hábitat y ecología. ........................................................................................ 12 2.1.4. Importancia. .................................................................................................. 13 2.1.4.1. Importancia medicinal. ............................................................................ 13 2.1.4.2. Importancia por su aprovechamiento maderable. ................................... 15 2.1.4.3. Importancia por su uso potencial. ..........................................................
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