AGROLISTIC TRANSFORMATION OF PLANT-CELLS - INTEGRATION OF T-STRANDS GENERATED IN PLANTA

We describe a novel plant transformation technique, termed ''agrolisti c,'' that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without unde sired vector sequence. The virulence genes virD1 and virD2 from Agroba cterium tumefaciens that are required in bacteria for excision of T-st rands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agroba cterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions wi th plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to pl ant cells) insertion events. We designate these as ''agrolistic'' inse rts, as distinguished from ''biolistic'' inserts. Both types of insert s were found in some transformed lines. The frequency of agrolistic in serts was 20% that of biolistic inserts.