TRANSPOSITION OF THE MAIZE AUTONOMOUS ELEMENT ACTIVATOR IN TRANSGENICNICOTIANA-PLUMBAGINIFOLIA PLANTS

The maize autonomous transposable element Ac was introduced into haplo id Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformat ion of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance g ene used to select primary transformants. The Ac excision frequency wa s determined using the phenotypic assay of restoration of neomycin pho sphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistan t seedlings in selfed progeny at a high frequency (34 to 100%) and con tained one or more transposed Ac elements. In the primary transformant s Ac transposition probably occurred during plant regeneration or earl y development. Other R0 transformants segregated kanamycin-resistant p lants at a low frequency (less-than-or-equal-to 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resis tant progeny would result from independent germinal transposition even ts. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2) , selected after respectively one or two selfings of these primary tra nsformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from on e to six and almost all transposition events were independent. Souther n analysis of the R2 and R3 progeny of these kanamycin-resistant plant s showed that Ac continued to transpose during four generations, and i ts activity increased with its copy number. The frequency of Ac transp osition, from different loci, remained low (less-than-or-equal-to 7%) from R0 to R3 generations when only one Ac copy was present. The strat egy of choosing R0 plants that undergo a low frequency of germinal exc ision will provide a means to avoid screening non-independent transpos itions and increase the efficiency of transposon tagging.