Análisis de resistencia y caracterización morfoanatómica de mutantes de zarzamora (Rubus fruticosus Cv. Tupy) obtenidas por radiación gamma, resistentes a Botrytis cinerea

Abstract

In order to reduce the use of synthetic fungicides for the control of the fungus Botrytis cinerea, which causes gray mold in a large number of crops of agricultural importance, such as blackberry (Rubus fruticosus), modern biotechnology offers in vitro mutagenesis by gamma radiation as an alternative for the production of resistant plants to this fungus. In the present research, mutant in vitro blackberry plants (R. fruticosus Cv. Tupy), rfgum5, rfgum6 y rfgum17, were obtained from shoots irradiated with gamma radiation, potentially resistant to B. cinerea, so resistance tests were carried out on in vitro plants and grown both in greenhouse and in the field (macrotunnel), as well as morphoanatomical studies to detect possible phenotypic changes compared to non-mutated (non-irradiated) plants. Shoot tips of in vitro-micropropagated blackberry plants (Rubus fruticosus ‘Tupy’) were irradiated with five doses of Cobalt-60 gamma radiation (0, 15, 30, 45, and 60 Gy) and cultured on Murashige and Skoog basal medium containing 1.0 mg/L benzyladenine and 0.06 mg/L indole-3-butyric acid (MSB medium). After 28 days of culture, survival was evaluated to determine mean lethal dose (LD50), and 200 shoots were further irradiated at the determined LD50 (30.8 Gy). After 28 days, the surviving shoots were micropropagated on MSB medium for 60 days. Non-irradiated shoots were screened for the in vitro selection of resistant B. cinerea, exposing them to different concentrations of sterile culture filtrate of B. cinerea (0, 2, 4, 6, 8, and 10 g/L) for 28 days to determine mean lethal concentration (LC50), and the irradiated surviving shoots were further exposed to the determined LC50 (4.6 g/L).

Con fines de disminuir el uso de fungicidas sintéticos para el control del hongo Botrytis cinerea, causante del moho gris en un gran número de cultivos de importancia agrícola, como la zarzamora (Rubus fruticosus), la biotecnología moderna ofrece la mutagénesis in vitro por radiación gamma como una alternativa para la producción de plantas resistentes a este hongo. En la presente investigación se seleccionaron plantas mutantes in vitro de zarzamora (R. fruticosus Cv. Tupy) rfgum5, rfgum6 y rfgum17, a partir de brotes irradiados con radiación gamma, potencialmente resistentes a B. cinerea, por lo que se realizaron ensayos de resistencia en plantas in vitro y cultivadas tanto en invernadero como en campo (macrotúnel), así como estudios morfoanatómicos para detectar posibles cambios fenotípicos en comparación con plantas no mutadas (no irradiadas). Se irradiaron yemas apicales de plantas micropropagadas de zarzamora Cv. Tupy con cinco dosis de radiación gamma cobalto-60 (0, 15, 30, 45 y 60 Gy) y se cultivaron en medio basal Murashige y Skoog con 1 mg/L de benciladenina y 0.06 mg/L de ácido indol-3-butírico (medio MSB). Después de 28 días de cultivo, se evaluó la supervivencia para determinar la dosis letal media (DL50), y se irradiaron 200 brotes a la DL50 determinada (30.8 Gy). Después de 28 días, los brotes sobrevivientes fueron micropropagados en medio MSB durante 60 días. Para la selección de brotes resistentes a B. cinerea, se cultivaron brotes no irradiados por 28 días en diferentes concentraciones de filtrado de cultivo estéril de B. cinerea (0, 2, 4, 6, 8 y 10 g/L) para determinar una concentración letal media (CL50) de 4.6 g/L.