STABLE GENETIC-TRANSFORMATION OF ARABIDOPSIS-THALIANA BY AGROBACTERIUM INOCULATION IN PLANTA

Abstract

Stable genetic transformation of Arabidopsis thaliana was achieved by simple in planta inoculation of Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI121. The transformation procedure, which we call in planta transformation, involves severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites. On average, 5.5% of the newly formed shoots produced transformed progenies. These progenies (T-2 generation) contained T-DNA in the genome as examined by assaying the T-DNA encoded beta-glucuronidase and kanamycin resistance and by genomic Southern blot analysis, the copy number of the T-DNAs in the Arabidopsis genome being single (33%) or multiple. The genetic behavior of the transformants examined at the T-3 and T-4 generations or with the F-2 progenies of the outcrosses between transformants and wild-type plants showed that most of the inserted T-DNA are inherited in a Mendelian fashion. This procedure provides a new approach for simple and efficient transformation of A. thaliana, obviating the need for plant regeneration from tissue explants in vitro.