Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana

Gene tagging in Arabidopsis thaliana using the autonomous Ac (Activator) transposable element has so far been hampered by low frequencies of germinal transposition events. Here we describe a procedure by which the frequency of independent germinal reinsertions has been much improved by a process of long-term selection on kanamycin for the continued growth of tissues in which somatic excisions have occurred. Growth on artificial media increased the somatic excision frequency, and the long-term selection procedure channelled somatic transposition events into the germline. This resulted in an overall germinal excision frequency in the progeny of longterm selected plants of 15%, as confirmed by Southern blotting, with 63% of the plants bearing excision events having detectable reinsertions of the Ac element. This compares with a germinal excision frequency of approximately 1% when no long-term selection is employed. However, offspring from individual plants tended to have identical germinal Ac reinsertion patterns, thus the critical parameter for evaluating the system for tagging purposes is the frequency of individual plants yielding offspring with reinsertions, which was 64%. This high frequency, when coupled to the enhanced germinal transposition rate overall, easily allows the generation of a large population of plants with independent reinsertions.