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.

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.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

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Two genes of Agrobacterium tumefaciens encode enzymes that together produce indoleacetic acid (IAA). The first gene, iaaM, encodes tryptophan monooxygenase which converts tryptophan to indoleacetamide (IAM). The second gene, iaaH, encodes indoleacetamide hydrolase which converts IAM to IAA. We have engineered each of the two genes to be expressed at either high constitutive levels or in a tissue-specific manner. These chimeric Recent advances in plant transformation and regeneration technology make plants ideal subjects for studying the interactions of different organs and cell types in a developmental system. Plants consist of a large number of distinct tissues and organs. The coordination of these many tissue types as a unit and the continuous develop~ment of new organs requires a complex system of communication. Several classes of phytohormones, including the cytokinins and auxins, can greatly influence the patterns of differentiation. The ability to manipulate the relative levels of phytohormones and observe the consequent effects would be extremely useful in elucidating the roles of these compounds in the processes of differentiation.Agrobacterium tumefaciens is the causative agent of crown gall disease, a neoplastic growth that affects many dicotyledonous plant species. It has been demonstrated that Agrobacterium transfers a portion of its DNA, the T-DNA, to the plant where it is integrated into plant nuclear DNA (for a review, see Fraley et al. 1986). Expression of several genes in the T-DNA results tPresent address: