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Universidad Austral De Chile Facultad De Ciencias Escuela De Ciencias Universidad Austral de Chile Facultad de Ciencias Escuela de Ciencias Profesor Patrocinante: Dr. Peter Seemann Fahrenkrog Instituto de Producción y Sanidad Vegetal Facultad de Ciencias Agrarias ESTABLECIMIENTO IN VITRO DE TRES ESPECIES CHILENAS DEL GÉNERO LOMATIA (PROTEACEAE) Seminario de Graduación presentado como parte de los requisitos para optar al Grado de Licenciado en Ciencias Biológicas. KATHERINE ANDREA BARRIENTOS CULÚN VALDIVIA – CHILE 2013 2 A mi pequeña hermanita, que llegó a llenar de alegría esta etapa de mi vida. 3 AGRADECIMIENTOS En primer lugar agradecer a mi Profesor Patrocinante Don Peter Seemann F, por sus consejos, guías y compromiso, además de su confianza entregada en el desarrollo de este trabajo. A Judith Carrasco y Susana, sus conocimientos entregados fueron de gran utilidad en el desarrollo de este trabajo y gracias por ser tan lindas personas conmigo, estoy infinitamente agradecida. A mis padres, por enseñarme que en la vida hay que tener respeto, responsabilidades y perseverancia, por entregarme la educación para utilizarla como herramienta en mi futuro, pero principalmente por su amor y cariño incondicional, que a pesar que estuve lejos de mi casa por estos años, nunca sentí la ausencia de ustedes en mi día a día. Los amo. A mi familia, por cada energía, aliento, sonrisa y más de alguna cajita feliz que llegó de Chiloé, ustedes igual aportaron en esta etapa. Los quiero mucho A mis amigos, por sus consejos y compañía, ustedes saben que son como mis hermanos, los quiero mucho. Camila y Dexsi, ustedes estuvieron apoyándome desde el comienzo de este trabajo, sin sus acciones creo que el camino hubiera sido más pesado para llegar al final, estoy enormemente agradecida de ustedes. 4 ÍNDICE 1. Resumen…………………………………………………………………………………7 1.1 Summary……………………………………………………………………………..8 2. Introducción……………………………………………………………………………..9 2.1 Familia Proteaceae y género Lomatia……………………………………………9 2.2 Cultivo de tejidos vegetales y Micropropagación……………………………...14 2.3 Micropropagación en Proteaceae……………………………………………….20 2.4 Hipótesis…………………………………………………………………………...23 2.5 Objetivo General…………………………………………………………………..23 2.6 Objetivo específicos……………………………………………………………….23 3. Materiales y Métodos…………………………………………………………………25 4. Resultados……………………………………………………………………………..37 4.1 Porcentaje de contaminación…………………………………………………….37 4.2 Porcentaje de sobrevivencia……………………………………………………...42 4.3 Formación de callo………………………………………………………………...44 4.4 Porcentaje de brotes o germinación……………………………………………..45 4.5 Pardeamiento………………………………………………………………………46 5. Discusión……………………………………………………………………………….48 5.1 Porcentaje de contaminación…………………………………………………….48 5.2 Porcentaje de sobrevivencia total………………………………………………..49 5.3 Porcentaje de formación de callo………………………………………………...51 5.4 Porcentaje de germinación o formación de brotes……………………………..51 5 5.5 Pardeamiento………………………………………………………………………52 6. Conclusiones…………………..…………………………….…………………......….54 7. Literatura citada…………………………………………………………………...….56 Anexos………………………………………………………………………….…..…..63 6 ABREVIACIONES 2,4 D: Ácido 2,4 diclorofenoxiacético, hormona vegetal (auxina). AIB: Ácido indolbutírico, hormona vegetal (auxina). ANA: Ácido naftalénacético, hormona vegetal (auxina). BAP: Bencil amino purina, hormona vegetal. MS: Medio de cultivo formulado por Murashige y Skoog (1962). PVP: Polivinilpirrolidona, polímero soluble en agua. WPM: =Woody Plant Medium, medio de cultivo formulado por Lloyd y McCown (1981). 7 1. RESUMEN En la actualidad se ha desarrollado comercialmente el valor ornamental de las proteáceas, el gran problema que presenta esta familia se debe a que son plantas difíciles de propagar. El objetivo de este trabajo es establecer in vitro tres especies chilenas de Lomatia: Lomatia ferruginea, Lomatia dentata y Lomatia hirsuta. La recolección del material vegetal se llevó a cabo en las épocas de otoño y primavera, utilizando como explantes semillas, yemas y brotes. Posteriormente a los explantes se les realizó una desinfección vegetal para ser sembrados y ser llevados a la sala de incubación. Su evaluación se realizó a los 45 días. Los resultados para las tres especies de Lomatia en la época de otoño no fueron muy favorables debido a que los explantes presentaron pardeamiento. Sin embargo, las semillas de esta época obtuvieron un alto porcentaje de germinación. En primavera, las yemas presentaron pardeamiento, y solo en la especie de Lomatia dentata se observaron brotes de novo utilizando explantes de brotes abiertos. El establecimiento in vitro de las especies Lomatia ferruginea, Lomatia dentata y Lomatia hirsuta puede ser exitoso teniendo en consideración que cada especie requiere de diferentes protocolos de desinfección, ya que cada una tiene características y necesidades diferentes, por lo que se debe considerar tratamientos específicos para cada especie. El explante que se utiliza también juega un rol importante. Si usa semilla, estas se pueden establecer en la época de otoño sin problemas y en primavera se recomienda utilizar brotes abiertos. 8 1.1 Summary Nowadays, the ornamental value of Proteaceae has been developed commercially. However, the problem that this plant family presents is that they are hard to propagate. The aim of this research is to establish in vitro three Chilean species of Lomatia ferruginea, Lomatia dentata and Lomatia hirsuta. The collection of the plant material was done during autumn and spring, using seeds and buds as explants sources. After that, the explants were submitted to disinfection prior to establishment and incubation. The evaluation was done after 45 days. The results for the three Chilean species of Lomatia during autumn were not favorable as the explants presented browning. However, the seeds obtained a high level of germination in this season. During spring, the buds showed browning and only Lomatia dentata presented de novo shoots, which wer were observed when using open buds. In vitro establishment of species like Lomatia ferruginea, Lomatia dentata and Lomatia hirsuta can be successfully carried out, taken into consideration that each species requires different disinfection protocols, because each one has different characteristics and needs, so it is necessary to consider specific treatments for each species. The explant used also plays an important role, by use of seeds, these can be set in autumn without problems. In spring open shoots are recommended for establishment of in vitro cultures. 9 2. INTRODUCCIÓN En la actualidad se investigan muchos métodos para facilitar la propagación de plantas, debido a que esta técnica es de gran utilidad para la conservación de plantas nativas. Con el fin de desarrollar nuevos métodos para que la propagación obtenga éxito, aparece la técnica de micropropagación, la cual permite que a partir de plantas difíciles de propagar por sistemas tradicionales, se obtengan cultivos libres de virus e incremente el número de individuos para evitar el peligro y extinción de las especies. Las proteáceas pertenecen a especies leñosas de gran valor ornamental, además algunas presentan endemismos, lo que las transforma en especies potenciales para su propagación, con fines comerciales y de conservación ex situ. 2.1 La familia Proteaceae y el género Lomatia La familia Proteaceae se originó al sur del continente Gondwana, comenzando a separarse en la era Mesozoica. Como consecuencia de este hecho, esta familia se encuentra confinada en la parte sur de los continentes (Kenneth y Criley, 1999). Existen cerca de 1400 especies, distribuidas en más de 60 géneros con dos subfamilias: Proteoideae, originaria principalmente del sur de África, pero también de Australia y Nueva Zelanda; Grevilleoideae, originaria de Australia y Sudamérica (Schiappacasse et al. 2006). Las proteáceas están conformadas por plantas leñosas, esclerófilas, polinizadas por aves y marsupiales (Strasburger et al., 1994). Su hábito estructural es variable a partir de las formas que presenta la cubierta del suelo, desde tallos rastreros a tallos subterráneos, también en la forma de crecimiento, variando desde arbustos 10 hasta árboles. Las hojas son generalmente lignificadas, duras y coriáceas. La anatomía de la hoja está especialmente adaptada a la conservación del agua, la resistencia a la sequía y daño por frío. Algunas presentan lignotúber (i.e., engrosamiento en la base de la planta, del cual pueden brotar yemas), raíces proteoídeas (i.e., racimos de raíces modificadas que absorben agua y nutrientes) e inflorescencias (i.e., dependiente del género, se pueden presentar en espigas, racimos, panículas, umbelas o capítulos) (Kenneth y Criley, 1999; Schiappacasse et al. 2006). Chile tiene 6 representantes, distribuidos en 4 géneros: Gevuina avellana (Avellano), Embothrium coccineum (Notro), Lomatia ferruginea (Fuinque), Lomatia dentata (Avellanillo), Lomatia hirsuta (Radal), Orytes myrtoidea (Radal Enano) (Donoso y Escobar, 1986). Dentro de la familia de las proteáceas, se encuentra el género Lomatia, el que se distribuye geográficamente en todas las áreas costeras al Océano Pacifico, desde Australia hasta Sudamérica. El hábitat de este género se presenta en climas templados, tropicales y subtropicales. En Sudamérica predomina el clima templado principalmente en bosques mediterráneos, con especies que presentan características de árboles pequeños y arbustos altos (Johnson y Briggs, 1975). Las tres especies de Lomatia presentes en Chile, corresponden a especies latifoliadas (especies de hojas anchas), cuyas características generales son, que poseen óvulos encerrados
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