Concentración mínima inhibitoria de higromicina B en callos embriogénicos de arroz -Oryza sativa L / Minimum inhibitory concentration of higromycin B in rice embryogenic calli -Oryza sativa L

Uno de los elementos imprescindibles en la ingeniería genética de plantas es un sistema de selección eficiente. El propósito de este trabajo fue evaluar la sensibilidad al marcador de selección higromicina B, de callos embriogénicos obtenidos a partir del escutelo de semilla de tres variedades colombianas de arroz (FEDEARROZ 2000, FEDEARROZ 50 y FEDEARROZ 369). Además, se validó la respuesta de estas variedades al protocolo de regeneración empleado. Se probaron cuatro concentraciones del antibiótico (25 mg/L, 50 mg/L, 75 mg/L y 100 mg/L) más un control sin higromicina B. Los resultados obtenidos mostraron que una concentración de 50 mg/L de antibiótico en el medio de regeneración es adecuada para la selección. Con esta concentración se impide la formación de brotes, aunque los callos no mueren completamente. Por otra parte, se estableció que el protocolo de regeneración utilizado es de baja eficiencia y, por consiguiente, es necesario optimizarlo para poder usarlo en procesos de ingeniería genética de cultivares colombianos de arroz.

An efficient selection system is one of the most important elements of plant genetic engineering. The purpose of this study was to evaluate the sensitivity of scutellum-derived embriogenic calli obtained from three colombian rice varieties (FEDEARROZ 2000, FEDEARROZ 50 and FEDEARROZ 369), to the selection marker hygromycin B. Aditionally, the response of these varieties to the regeneration protocol was measured. Four antibiotic concentrations were tested (25 mg/L, 50 mg/L, 75 mg/L and 100 mg/L) plus one control without hygromycin B. The results show that 50 mg/L of antibiotic in the regeneration medium is adequate for selection. This concentration prevents the formation of shoots, though the calli do not die. It was also established that the regeneration protocol is a low-efficiency system and it needs to be improved, in order to use it for colombian rice genetic engineering.

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.