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MICROPROPAGACIÓN in Vitro DE CACTUS COLA DE RATA

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MICROPROPAGACIÓN in Vitro DE CACTUS COLA DE RATA 0,&523523$*$&,Ï1LQYLWUR'(&$&786&2/$'(5$7$ $SRURFDFWXVIODJHOOLIRUPLV / 0$5Ë$0Ï1,&$52&+$&$,&('2 81,9(56,'$''(/72/,0$ )$&8/7$''(&,(1&,$6 352*5$0$'(%,2/2*Ë$ ,%$*8( 0,&523523$*$&,Ï1LQYLWUR'(&$&786&2/$'(5$7$ $SRURFDFWXVIODJHOOLIRUPLV / 0$5Ë$0Ï1,&$52&+$&$,&('2 7UDEDMRGHJUDGRSDUDRSWDUDOWtWXORGH%LyORJR 'LUHFWRU ',$1$0$5&(/$%(/75$13('52=$ 0VF&LHQFLDV%LROyJLFDV 81,9(56,'$''(/72/,0$ )$&8/7$''(&,(1&,$6 352*5$0$'(%,2/2*Ë$ ,%$*8( ADVERTENCIA La Facultad de Ciencias de la Universidad del Tolima, el director del trabajo y el jurado calificador, no son responsables de los conceptos ni de las ideas expuestas por el autor del presente trabajo. Artículo 16, Acuerdo 032 de 1976 y Artículo 29, acuerdo 064 de 1991, Consejo Académico de la Universidad del Tolima. 3 A mis padres, que me dieron la vida y han estado conmigo en cada momento. Ustedes lo han dado todo por mí y este trabajo no es más que una retribución a todo su esfuerzo. A mi hermana por su apoyo, confianza y amor. A la memoria de un hombre maravilloso: tú mi hermano del alma, aunque ya no estás entre nosotros, vives en mi corazón en cada momento, guiándome y acompañándome siempre. A ti mi hermosa Luisa María, la razón por la cual siempre deseo ser mejor día tras día. A mi amigos, compañeros de mil batallas. 4 AGRADECIMIENTOS Agradezco a Dios por estar conmigo en cada momento, especialmente en los difíciles. A Diana Marcela Beltrán, directora de este trabajo por haberme brindado todas las facilidades necesarias para realizar este trabajo en el laboratorio de protección de plantas y en especial por su constante impulso y colaboración en el desarrollo del mismo. Al profesor Neftalí Mesa por su apoyo en el desarrollo y finalización de este trabajo. A cada uno de los integrantes del grupo GEBIUT de la universidad del Tolima que de una u otra manera contribuyeron en la realización de este trabajo. 5 GLOSARIO ANTIOXIDANTE: es una molécula capaz de retardar o prevenir la oxidación de otras moléculas. ASÉPTICO: libre de patógenos contaminantes. AUXINAS: grupo de fitohormonas que funcionan como reguladoras del crecimiento vegetal. Esencialmente provocan la elongación de las células. CALLOGENESIS: proceso en el que a partir de un explante se genera una masa amorfa de células llamadas callo. DESINFECTANTE: sustancia que neutraliza y controla el proceso de contaminación y actúa sobre esta química o mecánicamente EXPLANTE: el explanto es un pequeño fragmento de una planta que se escinde y se prepara de forma aséptica para su cultivo en un medio nutritivo y que, por ende, funciona como generadora de nuevas plantas a través de cultivo de tejidos in vitro. MEDIO DE CULTIVO: un medio de cultivo puede ser definido como una formulación de sales inorgánicas y compuestos orgánicos requeridos para la nutrición y manipulación de las plantas a multiplicarse por cultivo in vitro. ORGANOGENESIS: proceso de diferenciación por el cual órganos de las plantas son formados de nuevo o desde estructuras preexistentes. OXIDACIÓN DE EXPLANTES: ennegrecimiento de los explantes debido a la exudación de productos químicos tales como fenoles y taninos. PLANTULAS: plantas pequeñas en proceso de desarrollo. REGULADOR DE CRECIMIENTO: sustancias organicas sintetizadas por la planta que intervienen en diversos procesos fisiologicos. TOTIPOTENTE: una característica celular, en la cual se conserva la potencialidad de formar todo tipo de células del organismo adulto. 6 RESUMEN Aporocactus flagelliformis L, es un cactus epifítico perteneciente a la familia Cactaceae, proveniente del norte de México. Puede encontrarse tanto en zonas desérticas como semidesérticas, soportando temperaturas extremas, es de uso potencial en la industria medicinal, en el forrajeo, en la industria cosmetológica, entre otras. Dentro del diario oficial de la federación Mexicana está considerada como una especie endémica; es atractiva a nivel ornamental por su forma de crecimiento, consistencia y belleza de sus flores. La dificultad en la adquisición de sus semillas y su alto costo condujo a la implementación de técnicas de cultivo de tejidos in vitro para una propagación más eficiente. En este proyecto se plantearon protocolos que permitieron el establecimiento del cultivo in vitro de la especie. Se evaluó el tipo de explante, agentes desinfectantes, antioxidantes y reguladores de crecimiento vegetal. Cortes transversales del domo apical central de tallo, con longitud mayor a 1 cm sin espinas, fue seleccionado como el explante óptimo para la propagación; para control de la contaminación se seleccionó una mezcla de yodo + antifúngico (Benomil, 3 gr/500 ml) en la etapa de acondicionamiento de plantas madres y el dicloruro de mercurio (HgCl2) al 0,2 % por 20 minutos, en la camara de flujo laminar. Como medio de cultivo se recomienda el MS básico, adicionado con 3 gr/L de carbón activado, BAP 3ml/L y ANA 1 ml/L. Con este protocolo se logró el control de la contaminación, la reducción en el porcentaje de oxidación de explantes, el establecimiento y propagación in vitro de A. flagelliformis L mediante organogénesis directa. Palabras clave: Aporocactus flagelliformis, oxidación, bencilaminopurina, ácido naftalenacético, organogénesis directa, carbón activado. 7 ABSTRACT Aporocactus flagelliformis L, is a cactus owned to the family Cactaceae, from northern México. It can be found both desert as semi-desert areas, withstanding extreme temperatures, is of potential use in the medical industry, the foraging, in the cosmetics industry, inter alia. Within the official journal of the Mexican federation is considered as a specie endemic; is attractive at level ornamental by his form of growth, consistency and beauty of its flowers; the difficulty in the acquisition of seeds and its high cost, make necessary the implementation of techniques of cultivation in vitro for a propagation more efficient. In this project were raised protocols which allowed the establishment of cultivation in vitro of the specie. Was evaluated the kind of explant, disinfectant agents, antioxidants and growth regulators. cross sections of dome central apical of stalk, with length higher of 1 cm boneless, was selected as the explant optimal for the propagation; for the control of the contamination, we selected a mixture of iodine + antifungal (Benomyl, 3 gr/500 ml) in the phase of conditioning of Mother plants and the mercury dichloride (HgCl2) to 0.2% for 20 minutes in the laminar flow chamber. was used as culture medium basic MS, suplemented with 3g/L of activated charcoal, 3 ml/L of benzyl aminopurine (BAP) and naphthaleneacetic acid 1ml/L. With this protocol was achieved the control of the contamination, the reduction in the percent of oxidation of explants, the establishment and propagation in vitro of A. flagelliformis L by direct organogenesis. Keywords: Aporocactus flagelliformis, oxidation, benzyl Aminopurine, naphthaleneacetic acid, direct organogenesis, activated charcoal 8 CONTENIDO Pág. INTRODUCCIÓN ................................................................................................... 14 1.OBJETIVOS ....................................................................................................... 16 1.1 GENERAL ........................................................................................................ 16 1.2 ESPECIFICOS ................................................................................................. 16 2. MARCO REFERENCIAL .................................................................................. 17 2.1 ANTECEDENTES ............................................................................................ 17 2.1.1 Familia Cactaceae ........................................................................................ 17 2.2 MARCO TEORICO .......................................................................................... 18 2.2.1 Características de familia Cactaceae ............................................................ 18 2.2.1.1 Origen y evolución ..................................................................................... 18 2.2.1.2 Distribución de las Cactáceas .................................................................... 18 2.2.1.3 Clasificación Botánica ................................................................................ 18 2.2.1.4 Morfología de las Cactáceas ...................................................................... 19 2.2.1.5 Fisiología de las Cactáceas ....................................................................... 22 2.2.1.6 Métodos de propagación............................................................................ 23 2.2.1.7 Amenaza y conservación ........................................................................... 24 9 2.3 DESCRIPCIÓN DE LA ESPECIE EN ESTUDIO ............................................. 24 2.3.1 Aporocactus flagelliformis L .......................................................................... 24 2.3.1.1 Clasificación ............................................................................................... 24 2.3.1.2 Descripción morfológica ............................................................................. 25 2.3.1.3 Distribución en México ............................................................................... 26 2.3.1.4 propagación de A. flagelliformis L .............................................................. 26 2.3.1.5 Conservación y factores de riesgo ............................................................. 26 2.4 CULTIVO in vitro DE TEJIDOS
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