Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable to plant species that can be infected by Agrobacterium and regenerated from leaf explants.

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We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.Agrobacterium tumefaciens is the etiologic agent of crown gall. Oncogenic strains of A. tumefaciens harbor large plasmids called Ti (tumor-inducing) plasmids (54-56). Part of the Ti plasmid-the transferred DNA (T-DNA)-is transferred to the plant, where it is stably maintained in the nuclear DNA (9, 10). The T-DNA contains genes that are transcribed in the plant (8), encoding enzymes for opine synthesis (5, 21, 38, 45, 51) and phytohormone synthesis (1,3,50,53). A second region of approximately 30 to 40 kilobase pairs (kbp) of the Ti plasmid that is involved in tumorigenesis but is not maintained in the tumor is called the virulence (vir) region. Drummond and Chilton (17) found extensive regions of DNA that were conserved on several wide-host-range Ti plasmids including octopine, nopaline, and L,L-succinamopine types. In an elegant study, Engler et al. (18) precisely defined these areas of homology between the vir regions of octopine and nopaline Ti plasmids. The vir regions from wide-host-range octopine (26,36,39,40,48) and nopaline (32, 43) Ti plasmids, as well as from a narrow-host-range octopine Ti plasmid (41), have been studied by mutagenesis and genetic complementation. The pTiC58 (nopaline) vir region contains six genetically defined complementation groups, one of which is cis dominant (43). Klee et al. (40) virulence genes for cross-functionality in a study using this binary system. Octopine T-DNA could be transferred to a variety of plants by the virulence regions of a root-inducing plasmid, a limited-host-range plasmid, and a wild-type nopaline plasmid, but not by Rhizobium sp. symbiotic plasmids. Otten et al. (48) showed by transient octopine assays that mutations in the virE locus of pTiB6S3 can be complemented by coinfection with wild-type Agrobacterium strains, consistent with the view that the product(s) of the virE locus must be diffusible.We report here a study of the hypervirulence of A. tumefaciens A281 on solanaceous plants. Our ...

Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 ,jg/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.

The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.