Optimización de la transformación genética vía Agrobacterium tumefaciens (Smith y Townsend) Conn en Phaseolus vulgaris L.
Fecha
2017-07-06
Autores
Ruíz de León, Lyselle
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Editor
Universidad Central “Marta Abreu “ de Las Villas. Facultad de Ciencias Agropecuarias. Departamento de Biología
Resumen
El frijol común (Phaseolus vulgaris L.), es una leguminosa de gran importancia económica a
nivel mundial. Su producción está limitada debido a factores bióticos y abióticos. La
transformación genética brinda la posibilidad de vencer estas limitaciones, pero la baja
eficiencia en la regeneración in vitro y la transferencia de ADN han limitado el desarrollo de
protocolos para la obtención de plantas transformadas en esta especie. El objetivo de este
trabajo fue la optimización de la transferencia de ADN de Agrobacterium tumefaciens a
P. vulgaris, cultivares 'ICA Pijao' y 'BAT 93'. Para ello, se determinó la utilización de ½ NC-1
como posible explante inicial para la regeneración vía organogénesis indirecta. La respuesta de
este explante se comparó con los explantes NC-1 y NC-2, utilizados en investigaciones previas.
También, se determinó el efecto de la concentración de la suspensión de A. tumefaciens, el
tiempo de infiltración por vacío y co-cultivo en la regeneración de brotes de P. vulgaris. El
empleo de ½ NC-1 como explante inicial para la regeneración de P. vulgaris, no afectó la
eficiencia del protocolo establecido para NC-1 y NC-2. La regeneración de los brotes, en los
cultivares 'BAT 93' e 'ICA Pijao', estuvieron influenciados por la concentración de la suspensión
de A. tumefaciens, el tiempo de infiltración por vacío y el período de co-cultivo. Los mayores
porcentajes de regeneración se lograron al inocular los callos con una concentración de la
suspensión de A. tumefaciens a DO600nm de 0,1, infiltrados con vacío por 1 o 2 min y
manteniéndolos tres días en co-cultivo a 22 ± 2°C.
Common bean (Phaseolus vulgaris L.) is a legume of great economic importance worldwide. Its production is limited due to biotic and abiotic factors. The genetic transformation offers the possibility of overcoming these limitations, but the low efficiency of in vitro regeneration and DNA transfer have limited the development of protocols for obtaining plants transformed in this specie. The aim of this work was to optimize the DNA transfer of Agrobacterium tumefaciens to P. vulgaris, 'ICA Pijao' and 'BAT 93' cultivars. For this, it was determined whether the ½ NC-1 can be used as the initial explant for regeneration via indirect organogenesis. The response of this explant was compared with NC-1 and NC-2 explants, used in previous research. In addition, the effect of the concentration of A. tumefaciens suspension, vacuum infiltration time and coculture in the regeneration of P. vulgaris shoots was determined. The use of ½ NC-1 as the initial explant for regeneration of P. vulgaris did not affect the efficiency of the established protocol for NC-1 and NC-2. The shoots regeneration, in cultivars 'BAT 93' and 'ICA Pijao', were influenced by the concentration of the A. tumefaciens suspension, the vacuum infiltration time and the co-culture period. The highest percentages of regeneration were achieved by inoculating callus with a concentration of the A. tumefaciens suspension to DO600nm of 0,1, vacuum infiltrated for 1 or 2 min and maintaining for three days of co-culture at 22 ± 2°C.
Common bean (Phaseolus vulgaris L.) is a legume of great economic importance worldwide. Its production is limited due to biotic and abiotic factors. The genetic transformation offers the possibility of overcoming these limitations, but the low efficiency of in vitro regeneration and DNA transfer have limited the development of protocols for obtaining plants transformed in this specie. The aim of this work was to optimize the DNA transfer of Agrobacterium tumefaciens to P. vulgaris, 'ICA Pijao' and 'BAT 93' cultivars. For this, it was determined whether the ½ NC-1 can be used as the initial explant for regeneration via indirect organogenesis. The response of this explant was compared with NC-1 and NC-2 explants, used in previous research. In addition, the effect of the concentration of A. tumefaciens suspension, vacuum infiltration time and coculture in the regeneration of P. vulgaris shoots was determined. The use of ½ NC-1 as the initial explant for regeneration of P. vulgaris did not affect the efficiency of the established protocol for NC-1 and NC-2. The shoots regeneration, in cultivars 'BAT 93' and 'ICA Pijao', were influenced by the concentration of the A. tumefaciens suspension, the vacuum infiltration time and the co-culture period. The highest percentages of regeneration were achieved by inoculating callus with a concentration of the A. tumefaciens suspension to DO600nm of 0,1, vacuum infiltrated for 1 or 2 min and maintaining for three days of co-culture at 22 ± 2°C.
Descripción
Palabras clave
Frijol, Phaseolus vulgaris L, ADN