Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium- mediated transformation-treated soybean cotyledons K.R. Finer 1 and J.J. Finer 2 1 Department of Biological Sciences, Kent State University/Stark Campus, Canton and 2 Department of Horticulture and Crop Sciences, OARDC, The Ohio State University, Wooster, OH, USA 2322/99: received 31 August 1999 and accepted 7 February 2000 K.R. FINER AND J.J. FINER. 2000. Colonization and infection of soybean cotyledons by Agrobacterium tumefaciens and subsequent elimination of bacteria from cotyledons were monitored using bacteria expressing green £uorescent protein (GFP). GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells. GFP was ¢rst detected 7 h following inoculation of the cotyledon. By 36 h, GFP expression was very intense, and was limited to the adaxial surface of the cotyledon. Expression of GFPalso served as a useful indicator of successful elimination of the bacterium from plant tissue following antibiotic treatment. INTRODUCTION Agrobacterium tumefaciens is a Gram-negative, soil-borne plant pathogen that has the ability to insert foreign genes into plant cells which then express the gene products. One major disadvantage of using Agrobacterium for plant transformation is the organism's host speci¢city, resulting in low levels of transformation in certain plant species.To increase transfor- mation e¤ciencies in these species, much e¡ort has been placed on understanding the molecular mechanisms of T- DNA transfer (Holford et al. 1992; Fullner et al . 1996) with the goal of manipulating and controlling the transfer process. Although these studies have been useful and have led to enhanced transformation rates in many plants, there have been few studies on the dynamics and optimization of agro- bacterial colonization and infection, the initial events upon which gene transfer between plant and host are ultimately dependent. The use of green £uorescent protein (GFP) from the jelly- ¢sh (Aequorea victoria) now provides us with a tool for moni- toring pathogen infection in time and space without disturbing either the bacterium or the host tissue. As no manipulation of the tissue is required for GFP visualization, the integrity of the cell structure and the morphology of the target tissue are maintained. In addition, because visualiza- tion of GFPdoes not kill cells, the £uorescence can be used to study the timing of gene expression in vivo (Chal¢e et al. 1994), or to monitor viral or bacterial infection events over time (Baulcombe et al. 1995; Gage et al. 1996; Oparka et al. 1997; Verver et al . 1998). By incorporating a plasmid into Agrobacterium tumefaciens that expresses GFP under the con- trol of a constitutive bacterial promoter, we were able to monitor Agrobacterium growth, on the surface of and within soybean cotyledons, over time under di¡erent cultural condi- tions. Soybean cotyledons were selected as the target tissue in this study as they are capable of producing somatic embryos, which are preferred for soybean transformation (Finer and McMullen1991). MATERIALS AND METHODS Bacterial strains and growth conditions The plasmid pTB93F (Gage et al. 1996; kindly provided by Sharon Long, Stanford University), encoding GFP was intro- duced into competent Agrobacterium tumefaciens EHA 105 (Hood et al. 1993; kindly provided by Beth Hood, Prodigene Inc., College Station, TX, USA) using a freeze/thaw method as previously described (Hofgen and Willmitzer 1988). Transformants which grew on modi¢ed Luria^Bertani (LB) agar (Trick and Finer 1997) supplemented with 300 mg ml 1 spectinomycin (Sigma, St Louis, MO, USA) were screened for GFP expression with a Leica M8 stereo dissecting micro- scope equipped with a £uorescence module consisting of a 100-W mercury lamp and GFP Plus 2 excitation and emis- Correspondence to: K.R. Finer, Kent State University, 6000 Frank Ave N.W., Canton, OH 44720, USA (e-mail: k®ner@stark.kent.edu). Letters in Applied Microbiology 2000, 30, 406410 = 2000 The Society for Applied Microbiology