Trabajo Final Conrado Pakoca

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Trabajo Final Conrado Pakoca UNIVERSIDAD NACIONAL DE LOMAS DE ZAMORA MAESTRIA EN FLORICULTURA "Bases para un programa de mejoramiento genético en Alstroemeria" Tesis presentada por el Ing. Agr. Conrado A. Pakoca para optar por el título de Magister en Floricultura 2014 AGRADECIMIENTOS Al Instituto de Floricultura CIRN INTA A la Cooperativa Argentina de Floricultores Ltda ÍNDICE GENERAL Resumen 1 Abstract 3 1 Introducción 5 1.1 Floricultura en Argentina ........................ 5 1.2 Historia del cultivo de Alstroemeria en Argentina ........... 6 1.3 Sistemática y distribución geográfica del género Alstroemeria . 11 1.4 Morfología y biología floral ....................... 13 1.5 Cariotipo ................................. 16 1.6 La hibridación interespecífica y el rescate de embriones como es- trategia del mejoramiento de Alstroemeria . 19 2 Objetivos 25 2.1 Objetivo general ............................. 25 2.2 Hipótesis ................................. 25 2.3 Objetivos específicos .......................... 25 3 Materiales y Métodos 27 3.1 Material vegetal ............................. 27 3.2 Evaluaciones preliminares ....................... 29 3.2.1 Estimación de la viabilidad del polen . 29 3.2.2 Evaluación del crecimiento del tubo polínico . 30 3.3 Cruzamientos dirigidos ......................... 32 3.3.1 Crecimiento del ovario post polinización . 32 3.4 Rescate de óvulos in vitro ........................ 33 3.4.1 Caracterización del crecimiento de los óvulos in vitro . 34 3.4.2 Desarrollo de brotes, raíces y aclimatación . 35 3.4.3 Coeficiente de eficiencia reproductiva . 35 3.5 Estimación del contenido relativo de ADN por citometría de flujo . 36 3.6 Registro ................................. 37 4 Resultados y Discusión 39 4.1 Evaluaciones Preliminares ....................... 39 4.1.1 Estimación de la viabilidad del polen . 39 4.1.2 Evaluación del crecimiento del tubo polínico . 40 i 4.2 Cruzamientos dirigidos ......................... 44 4.2.1 Crecimiento del ovario post polinización . 44 4.3 Rescate de óvulos in vitro ........................ 45 4.3.1 Caracterización del crecimiento de los óvulos in vitro . 45 4.3.2 Germinación in vitro ....................... 49 4.3.3 Análisis conjunto del crecimiento de los ovarios post polini- zación, caracterización de los óvulos, germinación in vitro y producción de plántulas .................... 52 4.3.4 Coeficiente de eficiencia reproductiva (CER) . 56 4.3.5 Desarrollo brotes, raíces y aclimatación . 59 4.4 Caracterización del contenido relativo de ADN mediante citometría de flujo .................................. 63 5 Conclusiones Generales 69 Bibliografía Consultada 71 ii INDICE DE TABLAS Tabla 1 Variedades y número de plantas introducidas en la primera importación de Alstroemeria para producción de flor de corte en la Argentina (1996) ...................... 7 Tabla 2 Variedades de Alstroemeria clasificadas por su porcentaje de participación y empresas fitomejoradoras presentes en el mer- cado a finales del año 2008. ................... 10 Tabla 3 Especies de Alstroemeria nativas de Argentina y su distribu- ción geográfica .......................... 13 Tabla 4 Especies, variedades y lugar de recolección de la colección de Alstroemeria del Instituto de Floricultura . 28 Tabla 5 Porcentaje de viabilidad del polen de las entradas de Alstroe- meria psittacina y las variedades comerciales (%) por el mé- todo de Greissl (1989) ...................... 40 Tabla 6 Germinación del polen y crecimiento del tubo polínico en cru- zamientos entre Alstroemeria psittacina, A. aurea y variedades comerciales. ............................ 42 Tabla 7 Volumen del ovario a 5 y 14 DPP (mm3) en cruzamientos en- tre especies y variedades de Alstroemeria: valores mínimos y máximos en cada grupo de cruzamientos . 47 Tabla 8 Crecimiento de los óvulos (ECO) a diferentes edades al res- cate (DPP) en diferentes combinaciones (%) . 48 Tabla 9 Germinación de óvulos (%) respecto al total de óvulos involu- crados en la polinización de todos los cruzamientos por com- binación. .............................. 51 Tabla 10 Combinaciones en las que se obtuvo germinación de embrio- nes en rescates realizados a diferentes tiempos post polinización 53 Tabla 11 Volumen teórico del ovario al rescate (VTOR) en mm3 a 14 DPP en ‘Belvedere’ × 20090602A4 y 'Belvedere' × 20091009B1 y sus recíprocos. 54 Tabla 12 Volumen teórico del ovario al rescate (VTOR) en mm3 de ‘Vir- ginia’ × 20091009B1 y ‘Virginia’ × 20100729C1 en rescates a 5 y 14 DPP en cruzamientos cuyos óvulos rescatados no germinaron ............................. 56 iii Tabla 13 Cruzamientos totales (Nº), cruzamientos que produjeron óvu- los que germinaron (Nº) y cruzamientos de los cuales se ob- tuvo plantas (Nº) para cada el estado de crecimiento del óvulo (ECO) observado a los 30 días de cultivo in vitro . 56 Tabla 14 Coeficiente de eficiencia reproductiva (CER) para ’Belvedere’, ‘Virginia’, 10#233 (A. aurea) y 10#263 (A. psittacina) en dife- rentes combinaciones. ...................... 58 Tabla 15 Número de cruzamientos, número de óvulos rescatados, nú- mero de embriones germinados, número de plantas obtenidas y porcentaje de plantas obtenidas respecto al número de óvu- los rescatados y al de óvulos germinados en rescates a 5 y 14 DPP ................................ 61 Tabla 17 Diferencia entre especies, variedades e híbridos respecto al trigo ‘Chinese Spring’ en el contenido relativo de ADN evalua- do por DAPI. ............................ 64 iv ÍNDICE DE FIGURAS Figura 1 Entrada 20081107I1, A. psittacina en el sitio de recolección Entrada a Santa Ana, Pcia. Corrientes, 27°27040:600 58°4101800 72 msnm. ............................. 27 Figura 2 Parcela de cultivo de variedades de Alstroemeria en el IF . 29 Figura 3 Cruzamientos identificados sobre Alstroemeria psittacina . 33 Figura 4 Medición de largo y ancho en ovarios de ‘Diamond’ en cruza- mientos con 20081107I1 (A. psittacina) rescatados a 5 DPP (168,169) y 20090602A4 (A. psittacina) en cruzamientos con ‘Belvedere’ rescatados a 14 DPP (149, 160) . 34 Figura 5 A Granos de polen viables (Vi) y no viables (NoVi) por el méto- do de Greissl correspondientes a 20090611Z1 (A. psittacina) B Granos de polen no viables de ‘Sacha’ . 41 Figura 6 Crecimiento de tubos polínicos A serpenteante y B errático del tubo polínico a 2 DPP en ‘Diamond’ × 20081107I1. C Tubos polínicos en la línea de abscisión del estilo, separado del ova- rio en ‘Sacha’ × 20081107I1. Barra=50 µm. 43 Figura 7 Crecimiento del tubo polínico a 2DPP, A terminaciones globo- sas anormales en la mitad del estilo, en ‘Sacha’ × 20081107I1 y B en ‘Fuego’ × 20090611Z1, C Crecimiento serpenteante del tubo polínico en el ovario con variación del diámetro en 20081107I1 × ‘Jive’ Barra=50 µm. 44 Figura 8 A Crecimiento serpenteante del tubo polínico en la zona mi- cropilar a 2 DPP en ‘Diamond’ × 20090602Z2. Barra=40 µm B Deposición de calosa en la región de la chalaza y funículo en ‘Fuego’ × ‘Belvedere’ Barra=200 µm. 45 Figura 9 Estado de crecimiento de los óvulos. (a) ECO 1, 2 y 3 (b) ECO 4 48 Figura 10 A Embrión germinado, 20091009B1(*) × ‘Belvedere’, resca- te a 14 DPP y 103 días de cultivo in vitro. (*) A. psittacina.B Embrión germinado, 20091009B1(*) × ‘Belvedere’, rescate a 19 DPP y 82 días de cultivo in vitro. (*) A. psittacina. C Em- brión germinado, 20090602A4(*) × 20081107I1(*), rescate a 25 DPP y 70 días de cultivo in vitro. (*) A. psittacina . 52 Figura 11 A Plántula, ‘Belvedere’×10#263(*), rescate a 8 DPP y 112 días de cultivo in vitro. (*) A. psittacina B Plántula, ‘Virginia’ × 20091009B1(*), rescate a 14 DPP y 105 días de cultivo in vitro. (*) A. psittacina. ....................... 60 v Figura 12 A Planta en aclimatación, ‘Virginia’ × 20091009B1(*), resca- te a 14 DPP 200 días de cultivo in vitro y 14 días en ma- ceta. (*) A. psittacina B Planta en aclimatación, ‘Virginia’ × 20091009B1(*), rescate a 14 DPP 227 días de cultivo in vitro y 137 días en maceta. (*) A. psittacina . 61 Figura 13 Flores de 20091009B1 (A. psittacina) (izquierda), 'Virginia' × 20091009B1 -cruzamiento 11-1052-(centro) y 'Virginia' (dere- cha) ................................. 65 Figura 14 Inflorescencias de 20091009B1 (A. psittacina) (izquierda), 'Vir- ginia' × 20091009B1 -cruzamiento 11-1052-(centro) y 'Virgi- nia' (derecha) ........................... 65 Figura 15 Contenido relativo de ADN obtenido por citometría de flujo de A 20091009B1 (A. psittacina) (1), ‘Virginia’×20091009B1- cru- zamiento 11-1051 (2) y ‘Virginia’ (3) . 66 Figura 16 Contenido relativo de ADN obtenido por citometría de flujo de 20091009B1 (A. psittacina) (1), ‘Virginia’ × 20091009B1, cru- zamiento 11-1052 (2), y ‘Virginia’ (3) . 67 Figura 17 Contenido relativo de ADN obtenido por citometría de flujo de 20091009B1 (A. psittacina) (1), ‘Belvedere’×20091009B1 cru- zamiento 11-1058 (2) y ‘Belvedere’ (3) . 68 vi LISTA DE ABREVIATURAS 1-NAA 2-(1-Naphthyl)acetic acid ó Acido 1-Naftalen Acetico ó Acido alfa naftalen acético 2-4D Acido 2,4-diclorofenoxiacético 6-BA N-benciladenina ó N-bencil-7H-purin-6-amina. Sin= BA, BAP ADN Acido desoxiribonucleico CAF Cooperativa Argentina de Floricultores Ltda. CER Coeficiente de Eficiencia Reproductiva CETEFFHO Centro Tecnológico en Floricultura Fruticultura y Hor- ticultura DAPI 2-(4-amidinofenil)-1H -indol-6-carboxamidina DPP Días Post Polinización ECO Estado de crecimiento del óvulo FAA Formol-Acido Acético-Alcohol FDA Diacetato de fluoresceina GA3 Acido Giberelico GISH Genomic In Situ Hybridization IBA Acido Indol 3-butírico INTA Instituto Nacional de Tecnología Agropecuaria MS Murashigue-Skoog
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