| Maize transposable elements, Activator (Ac) and Dissociation (Ds) have been transformed into several heterologous plant species for transposon tagging of genes and they have been successfully used to tag and to clone genes in Arabidopsis , flax, petunia, tobacco, and tomato. In developing an Ac /Ds based two-element transposon tagging system, transposition of Ds has been investigated in carrot ( Daucus carota L).; Carrot calli were transformed with Ds and Ac -TPase by using Agrobacterium tumefaciens strain LBA4404. Calli, initially transformed with Ac-TPase, were transformed for the second time with Ds. Southern analysis demonstrated that from one to at least nine copies of T-DNA inserted into the genome of carrot by Agrobacterium. Transposition of Ds was not detected in any of the calli or transgenic plants carrying Ds only. On the other hand, Ds excised in all of the callus lines and tissue culture-regenerated plants carrying both Ac-TPase and Ds. Reinsertion of Ds into new chromosomal sites was detected in transgenic plants based on Southern blotting and sequence analysis of Ds insertion sites using TAIL-PCR. Our results indicated that Ds will transpose in the carrot genome if Ac-TPase is present in the same nucleus.; Carrot plants carrying both Ac-TPase and Ds were also produced by crossing plants carrying Ac-TPase with plants carrying the Ds element. PCR and Southern analyses indicated that Ds did not excise in any of the T1 progeny plants carrying both Ac-TPase and Ds. RT-PCR analysis demonstrated that Ac-TPase gene was expressed in transgenic carrot plants carrying both Ac-TPase and Ds as well as those containing only Ac-TPase. Correctly spliced transcripts of introns 1, 2, 3 and 4 were produced although the presence of an incorrectly spliced product of intron 4 was also detected by RT-PCR. Excision of the Ds element from the original T-DNA construct was detected in calli which were initiated from T1 progeny plants carrying both Ac-TPase and Ds. These results suggested that Ds element can be reversibly inactivated in the somatic tissues of carrot, and this inactive Ds element can be mobilized in tissue culture. |
