Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA, but no Ri-plasmid T-DNA

Abstract

Cultivated tomato was genetically transformed using two procedures. In the first procedure, punctured cotyledons were infected with "disarmed" Agrobacterium tumefaciens strain LBA4404 or with A. rhizogenes strain A4, each containing the binary vector pARC8. The chimeric neomycin phosphotransferase (NPT II) gene on pARC8 conferred on transformed plant cells the ability to grow on medium containing kanamycin. Transformation reproducible yielded kanamycin-resistant transformants in different tomato genotypes. NPT II activity was detected in transformed calli and in transgenic plants. All of these plants were phenotypically normal, fertile and set seeds. Using the second procedure, inverted cotyledons, we recovered transformed tomato plants from A. rhizogenes-induced hairy roots. In this case, all of the transgenic plants exhibited phenotypes similar to hairy root-derived plants reported for other species. Southern blot analysis on these plants revealed that the plant DNA hybridized with both probes representing pARC8-T-DNA, and the T-DNAs of the A4 Ri-plasmid. However, southern analysis on those phenotypically normal transgenic plants from the first procedure revealed that only the pARC8-T-DNA was present in the plant genome, thus indicating that the pARC8-T-DNA integrated into the plant genome independently of the pRi A4-T-DNA. Genetic analysis of these phenotypically normal transgenic plants for the kanamycin-resistance trait showed Mendelian ratios, 3∶1 and 1∶1, for selfed (R1) and in crossed progeny, respectively.