Factors Affecting the Agrobacterium-Mediated Transient Transformation of the Wetland Monocot, Typha latifolia

Abstract

An Agrobacterium-mediated transformation system, using transient transformation assays, was used to evaluate conditions influencing transformation for the wetland monocot Typha latifolia. These studies were aimed at the long-term objective of evaluating candidate genes for phytoremediation. The binary plasmid vector pCAMBIA1301/EHA105, containing the β-glucuronidase coding sequence, was used in combination with factors known to affect transformation. These included callus age at the time of cocultivation with Agrobacterium tumefaciens, type and concentration of auxin for explant growth, light or dark culture environment, the presence or absence of acetosyringone (AS), explant type, explant wounding and the number of days used for cocultivation. The number of days needed for the first detection of transient expression of the β-glucuronidase gene was also examined. Three days of Agrobacterium cocultivation of 50-day-old seedling-derived calluses, grown on 20.7 µM (5 mg l−1) picloram supplemented medium, in the dark, resulted in higher levels of transient β-glucuronidase expression than were seen in calluses cultured on 4.5 or 22.6 µM (1 or 5 mg l−1) 2,4-dichlorphenoxyacetic acid containing media. The addition of 100 µM acetosyringone significantly enhanced transient β-glucuronidase activity. Wounding of explants, by cutting into two or three pieces, 3 days before cocultivation, increased expression of β-glucuronidase only in calluses cultured under light conditions. Transient β-glucuronidase expression was observed as early as 24 h after cocultivation and increased as the days post cultivation increased. The developed transient system will be used for stable transformation of Typha species.