AGROBACTERIUM-TUMEFACIENS TRANSFORMATION AND COTRANSFORMATION FREQUENCIES OF ARABIDOPSIS-THALIANA ROOT EXPLANTS AND TOBACCO PROTOPLASTS

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency o f transformation and cotransformation was determined during cocultivat ion of Arabidopsis thaliana root explants and Nicotiana tabacum protop lasts with two Agrobacterium strains. The transformation frequency of unselected A, thaliana shoots was lower than 1% whereas that of cocult ivated tobacco protoplasts was approximately 18%, The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approxima tely 40% reproducible, irrespective of the selection, the transformati on frequency, and the plant system used. Extrapolation of these result s suggests that at least two independently transferred T-DNAs were pre sent in 64% of the transformed plant cells, Molecular analysis of cocu ltivated N. tabacum shoots regenerated on nonselective medium showed t hat only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA, Therefore, most integrated T-DNAs expressed their selectable o r screenable markers in primary transgenic plants. Remarkably, 10 to 3 0% of the selected A. thaliana shoots or progenies lost the T-DNA mark er they were selected on. As these regenerants contained the unselecte d T-DNA with a high frequency (17%), these selected plants might resul t from the expression of unstable, transiently expressed T-DNAs, In co nclusion, a significant part of the T-DNAs is lost from the transforme d cells.