Estudio de parámetros involucrados en la transformación genética de la caña de azúcar (Saccharum spp) mediante embriogénesis directa
Palabras clave:
caña de azúcar, proteína verde fluorescente, embriones somáticos directos, micropartículas de oro, eficiencia de transformación, sugarcane, green fluorescent protein, direct somatic embryos, gold microparticles, tungsten microparticles, transformation efficiencyResumen
El tiempo promedio requerido para regenerar una planta transgénica completa a partir de callos embriogénicos (Embriogénesis Somática Indirecta, ESI) es de seis a nueve meses. La embriogénesis somática directa (ESD) es una vía morfogénica alternativa que permite obtener embriones somáticos directamente desde células aisladas o grupos de células sin la formación del callo, lo cual conlleva diversas ventajas tales como la disminución en el tiempo de regeneración, el menor número de subcultivos y una menor probabilidad de aparición de variantes somaclonales. Además, otros parámetros del proceso de transformación como tipo de microproyectiles, edad de los embriones somáticos y cantidad de ADN pueden afectar la eficiencia del mismo. El objetivo del presente trabajo fue estudiar la eficiencia de transformación genética de la variedad comercial local de caña de azúcar TUC 95-10 a partir de explantos obtenidos por ESD. Se ensayaron, asimismo, las variables tipo de micropartículas, edad de los embriones somáticos y cantidad de ADN mediante el uso del gen reportero gfp (del inglés “green fluorescent protein”),bajo la regulación del promotor constitutivo CaMV35S. El mayor número de puntos de expresión estables (puntos GFP) se observó cuando los bombardeos se efectuaron con partículas de oro. En la evaluación de las diferentes edades de los embriones somáticos (10 y 18 días)y concentraciones de ADN (2,5 y 5 μg) no se observaron diferencias significativas en el número de puntos GFP. Los resultados sugieren que la utilización de partículas de oro con una concentración de ADN de 2,5 μg y explantos de discos de hojas de 10 días mejora la eficiencia del protocolo de transformación genética en caña de azúcar, reduciendo el tiempo y la cantidad de ADN requeridos.
ABSTRACT
Study of the parameters involved in the genetic transformation of sugarcane(Saccharum spp) by direct embryogenesis
The average time required to regenerate a complete transgenic plant from embryogenic calli (Indirect Somatic Embryogenesis, ESI) is 6 to 9 months. Direct somatic embryogenesis (ESD) is an alternative morphogenic pathway that allows obtaining somatic embryos directly from isolated cells or groups of cells without the formation of callus, which has several advantages such as the reduction in regeneration time, the lower number of subcultures and a lower probability of somaclonal variation. In addition, other parameters of the transformation process such as the type of microprojectiles, the age of the somatic embryos and the amount of DNA can affect its efficiency. The objective of the present work was to study the genetic transformation efficiency of the local commercial sugarcane variety TUC 95-10 from explants obtained by ESD. Likewise, the variables type of microparticles, age of the somatic embryos and amount of DNA were analized by using the reporter gene gfp (“green fluorescent protein”), under the regulation of the constitutive promoter CaMV35S.
The highest number of stable expression points (GFP points) was observed when bombardments were carried out with gold particles. In the evaluation of the different ages of the somatic embryos (10 and 18 days) and DNA concentrations (2.5 and 5 μg), no significant differences were observed in the number of GFP points. The observed results suggest that the use of gold particles with a DNA concentration of 2.5 μg and explants of 10-day-old leaf discs improves the efficiency of the genetic transformation protocol in sugarcane.
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