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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:

SummaryThe visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum in®ltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per in®ltrated plant were achieved. The results showed that the majority of the transgenic seeds from single in®ltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the in®ltrated plants and the microscopic analysis of GUS expression in ovules of in®ltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-in®ltration. In addition, all ovules from a single in®ltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3±5 days postin®ltration, whereas frequencies of up to 6% were detected with ovules of unopened¯owers 5±11 days post-in®ltration. Most importantly, transgenic seeds were obtained only from genetic crosses using in®ltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

Since the first demonstration of GFP from the jellyfish Aequorea victoria as a vital reporter for gene expression in both bacteria and Caenorkabditis elegans (Chalfie et al., 1994), GFP has attracted widespread interest and is considered to have severa1 advantages over other visual marker genes. First, the fluorescence emission of GFP does not require a cofactor or a substrate; fluorescence results from an interna1 p-hydroxybenzylidene-imidazo-lidinone chromophore generated by cyclization and oxidation of a SerTyr-Gly sequence at amino acid residues 65 to 67 (Cody et al., 1993). Detection of GFP in living cells thus only requires excitation by light at 395 or 470 nm. In contrast, the assay of GUS expression is cytotoxic, firefly luciferase (Ow et al., 1986;Millar et al., 1995) requires an exogenous substrate (luciferin) for detection, and plant anthocyanins (Klein et al., 1989;Lloyd et al., 1992) are generally useful only in mature, differentiated cells.The second advantage of GFP is that it is relatively small (26.9 kD) and can tolerate both N-and C-terminal protein fusions, lending itself to studies of protein localization and intracellular protein trafficking (Wang and Hazelrigg, 1994;Davis et al., 1995;Kaether and Gerdes, 1995). Another advantage of GFP is that GFP mutants with shifted wave-

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