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Universitat Politècnica De València Universitat Politècnica de València Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural (ETSIAMN) Grado en Ciencia y Tecnología de los Alimentos Regeneración de plantas en cultivo in vitro de especies silvestres relacionadas con tomate Nicolás Gil Sepulcre Tutor: Alejandro Atarés Huerta Primer cotutor: Vicente Moreno Ferrero Directora experimental: Marybel Jáquez Gutiérrez Curso académico 2016/2017 València, junio 2017 Título: Regeneración de plantas en cultivo in vitro de especies silvestres relacionadas con tomate Resumen: El cultivo in vitro engloba un conjunto de técnicas que permiten la multiplicación del material vegetal, la mejora sanitaria y la mejora genética de plantas. Dentro de estos procesos se incluyen técnicas como la micropropagación de variedades de alto valor comercial, el cultivo de meristemos, el microinjerto, el incremento de la variabilidad genética; tanto intraespecífica mediante el uso de la variación somaclonal como extraespecífica mediante el rescate de embriones o la hibridación somática mediante fusión de protoplastos, la obtención de plantas haploides y doble-haploides y la transformación genética. El tomate es la especie hortícola más importante a nivel mundial después de la patata. Su consumo se ha extendido prácticamente por todo el mundo, tanto en fresco como en forma de salsas, zumos, concentrados, etc. Existen diversas especies silvestres relacionadas filogenéticamente que pueden servir como fuentes de variación genética en los programas de mejora de la especie cultivada. En nuestro laboratorio se han puesto a punto diversos métodos de regeneración mediante inducción de organogénesis adventicia de algunas especies silvestres relacionadas con tomate. Su aplicación en técnicas como la transformación genética nos ha permitido obtener un material muy valioso para la identificación de genes clave en múltiples procesos mediante un programa de mutagénesis insercional. En este contexto, la mejora de un método de regeneración o su puesta a punto, en el caso de trabajar con una nueva especie, puede abrir nuevas vías en las investigaciones que está llevando a cabo nuestro grupo. Teniendo en cuenta todo lo anterior, el objetivo central de este proyecto consiste en profundizar en el conocimiento de la inducción de organogénesis adventicia en Solanum lycopersicum var. cerasiforme, un ancestro del tomate cultivado y en Solanum pimpinellifolium y Solanum galapagense, dos especies silvestres del mismo género. Para ello se ha estudiado la influencia que tiene sobre estos genotipos diversos factores como el tipo de explante, el medio de cultivo y las condiciones de incubación sobre la inducción de organogénesis adventicia. El desarrollo de estos métodos tiene una gran importancia para su aplicación en programas de mejora basados en la inducción de variabilidad (i.e. variación somaclonal) o la transformación genética. Palabras clave: Morfogénesis - Cultivo in vitro – Tomate – Especies silvestres Autor: D. Nicolás Gil Sepulcre Localidad y fecha: València, junio 2017 Tutor Académico: Dr. Alejandro Atarés Huerta Cotutor: Dr. Vicente Moreno Ferrero Directora experimental: Marybel Jáquez Gutiérrez Tipo de licencia de autorización de acceso y difusión del TFG/M: Creative Commons: Reconocimiento – NoComercial (by-nc). Title: Plant regeneration by tissue culture in tomato wild related species Abstract: In vitro culture encompasses a set of techniques that allow the multiplication of plant material, vegetal material sanitation and genetic improvement of plants. These processes include techniques such as varieties of high commercial value, meristem culture, micropropagation of varieties of high commercial value, growing from meristems, the micrografts, genetic variability increased; both intraspecific using somaclonal variation and extraespecific by embryo rescue or somatic hybridization by fusion of protoplasts, obtaining haploid and doble-haploides plants and genetic transformation. The tomato is the most important horticultural species around the world after the potato. It´s use has spread practically by everyone, both fresh and in form of sauces, juices, concentrates, etc. There are several wild species phylogenetically related that may serve as sources of genetic variation improvement of the cultured species programs. In our laboratory we have done various methods of regeneration through induction of adventitious organogenesis of some wild species related to the tomato. It is the application in techniques like genetic transformation that has allowed us to obtain a very valuable material for the identification of key genes in multiple processes through a programme of insertional mutagenesis. In this context, the improvement of a method of regeneration or their implementation, in the case of work with a new species, can open new avenues in the investigation that our group is carrying out. Taking into account all of the above, the central objective of this project is to deepen the knowledge of the induction of adventitious organogenesis in Solanum lycopersicum var. Cerasiforme, an ancestor of the cultivated tomato and in Solanum pimpinellifolium and Solanum galapagense, two wild species of the same genus. In order to do so, we have studied the influence of different factors such as explant type, culture medium and incubation conditions on the induction of adventitious organogenesis. The development of these methods is of great importance for their application in improvement programs based on the induction of variability (i.e., somaclonal variation) or genetic transformation. Keywords: Morphogenesis – In vitro culture – Tomato – Wild species Agradecimientos Este trabajo no hubiese sido posible sin la ayuda de mucha gente. Me gustaría expresar mi agradecimiento a todas las personas que me han apoyado y a todo el grupo de investigación, en especial a Vicente Moreno por darme la oportunidad de formar parte de esa gran familia que es el laboratorio 0.07. Son muchas las personas implicadas en la consecución de este proyecto y me gustaría hacer una serie de menciones: Gracias a Alex por dirigir este trabajo y estar el día a día para cualquier duda. Gracias a Marybel por enseñarme a trabajar y a preparar café. A Jesús por estar siempre dispuesto a echar una mano y hacerlo. A toda mi familia en especial a mi madre y a mi hermano. A todos mis amigos y gente maravillosa que ha pasado por mi vida este año. A todos ellos, gracias. ÍNDICE GENERAL 1. Introducción .............................................................................................................................. 1 1.1. Origen, domesticación y clasificación taxonómica ............................................................ 1 1.2. Descripción botánica .......................................................................................................... 1 1.3. Importancia y relevancia científica .................................................................................... 2 1.4. Importancia económica ...................................................................................................... 2 1.5. Mejora genética de tomate ................................................................................................ 3 1.6. Fundamentos y aplicaciones del cultivo in vitro ................................................................ 5 1.7. Aplicaciones del cultivo in vitro a la mejora genética del tomate ..................................... 6 OBJETIVOS ..................................................................................................................................... 8 3. Materiales y Métodos ............................................................................................................... 9 3.1. Material vegetal ................................................................................................................. 9 3.2. Técnicas básicas de cultivo in vitro..................................................................................... 9 3.2.1. Esterilización de semillas ............................................................................................. 9 3.2.2. Preparación y esterilización de los medios de cultivo .............................................. 10 3.2.3. Trabajo en condiciones asépticas.............................................................................. 10 3.2.4. Germinación de semillas ........................................................................................... 10 3.2.5. Cultivo de explantes de cotiledón y de ápices meristemáticos ................................ 11 3.2.6. Cultivo de peciolo, peciolulo y limbo de hoja ........................................................... 11 3.2.7. Tipos de explantes ..................................................................................................... 11 3.3. Citometría de flujo ........................................................................................................... 13 3.3.1. Estudio del patrón polisomático del material de partida ......................................... 13 3.3.2. Estudio del nivel de ploidía de las plantas regeneradas ........................................... 13 3.4. Regeneración de plantas .................................................................................................. 14 3.4.1. Tipos de medio de cultivo ......................................................................................... 14 3.4.2. Evaluación de la regeneración .................................................................................
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