Transformation of tomato using an Ri plasmid vector

Abstract An intermediate vector, pAMNeo10, was constructed containing the replication origin and carbenicillin-resistance gene of pBR322, an homology region to allow insertion into the T L -DNA of pRiA4 in Agrobacterium A4T, and a chimaeric kanamycin-resistance gene ( nop. neoΔ ) for identification of T L -DNA::pAMNeo10 transformed roots. Roots produced by inoculating stem explants of Lycopersicon esculentum, L. hirsutum × L. esculentum (KNVF Rootstock) and L. peruvianum with an exconjugant stain, A4T (pRiA4::pAMNeo10), were resistant to kanamycin at levels that completely inhibit the growth of transformed roots produced with wild-type A4T. When transformed by the exconjugant strain, roots of the three tomato hosts were resistant to different levels of kanamycin, and, in the case of L. peruvianum , regenerated plants were tolerant to much higher levels (10×) of kanamycin than the transformed roots from which they were derived. Kanamycin-resistant transformed roots expressed aminoglycoside phosphotransferase activity, and Southern blotting confirmed the presence of the intermediate vector sequence in transformed roots and in shoots of regenerated plants. T R -DNA was shown to be present in most transformed roots and regenerated shoots by testing for agropine and mannopine. The application of Ri plasmid vectors to the study of foreign gene expression in plants is discussed.