Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential

Protocol and analysis of in vivo pollen tuve growth = Protocolo y análisis del crecimiento in vivo del tubo polínico en yuca/mandioca

This 9-minute video describes the protocol used in cassava for the analysis pollen tube grow in vivo. Then images of pollen tube growth taken with a fluorescence microscope are presented to illustrate the process. Finally an overall description of the time required for the pollen tube tip to reach the embryo sac is presented. Este video de 9 minutos describe el protocolo usado en yuca/mandioca para el análisis del crecimiento del tubo polínico in vivo. Luego se presentan numerosas fotografías, tomadas en microscopio de fluorescencia, para ilustrar el proceso hasta que el tubo polínico llega al saco embrionario. Finalmente se presenta información de los tiempos requeridos en todo el proceso

Protocol of ovule culture for rescuing embryos in cassava = Protocolo para el cultivo de óvulos en el rescate de embriones en yuca/mandioca

This 14-minute video describes the protocol used for the collection of female flowers at different ages after anthesis and the development of a protocol of early embryo rescue. The production of doubled haploids through different gynogenesis approaches usually require an early rescue of embryos which otherwise would die. Significant progress could be made and plants could be regenerated from embryos rescued less than 14 day after anthesis. The video presents the salient points of the protocols used as well as images illustrate the appearance of tissue during the process. Este video de 14 minutos describe el protocolo usado en la colección de flores femeninas de diferentes edades y el desarrollo del protocolo para el rescate temprano de embriones. La producción de doble haploides a través de distintas estrategias de ginogénesis generalmente requiere de un rescate temprano de embriones antes que ellos mueran. Se reporta un progreso significativo al regenerar plantas a partir de embriones que habían sido rescatados antes de los 14 días de la antesis. El video presenta la información más relevante del protocolo usado así como imágenes ilustrando los cambios en apariencia del tejido a lo largo del proceso

Vibratome technology for histological analysis of embryo development in cassava = Utilización del vibrátomo para análisis histológico del desarrollo embrionario en yuca/mandioca

This 9-minute video describes the protocol used in cassava for the analysis pollen tube grow in vivo. Then images of pollen tube growth taken with a fluorescence microscope are presented to illustrate the process. Finally an overall description of the time required for the pollen tube tip to reach the embryo sac is presented. Este video de 9 minutos describe el protocolo usado en yuca/mandioca para el análisis del crecimiento del tubo polínico in vivo. Luego se presentan numerosas fotografías, tomadas en microscopio de fluorescencia, para ilustrar el proceso hasta que el tubo polínico llega al saco embrionario. Finalmente se presenta información de los tiempos requeridos en todo el proceso

Protocol of anther and microspore culture in cassava (androgenesis) = Protocolo para el cultivo de anteras y microsporas (androgénesis) en yuca/mandioca

This 9-minute video describes the protocol used for the collection of male flowers in cassava for the production of doubled haploids through androgenesis (in vitro microspore or anther culture). Pre-treatments and different culture conditions used in the process to induce cell division in immature microspores are described. Progress inducing cell division and calli was made, but no plants could be regenerated. Este video de 9 minutos describe el protocolo usado en la colección de flores masculinas de yuca/mandioca para la producción de doble haploides a través de androgénesis (cultivo de microsporas o anteras). Se muestran algunos de los pre-tratamientos así como condiciones de cultivo in vitro para inducir división celular en las microsporas. Se describen los éxitos alcanzados induciendo división celular y producción de callos. No se pudo, sin embargo, regenerar plantas

Description of embryo development in cassava = Descripción del desarrollo embrionario en yuca

This 16-minute video describes the protocol used for the preparation of sections for the histological analysis of cassava pistils of different ages after anthesis. Consolidated results after studying hundreds of samples, present a chronological description of embryo sac and the embryo appearance at different stages of development. Este video de 16 minutos describe el protocolo usado en la preparación de muestras para el estudio histológico de pistilos de yuca/mandioca a diferentes edades luego de la antesis. Resultados consolidados, después de analizar cientos de secciones, presentan una descripción cronológica de los diferentes estadios de desarrollo embrionario