Mol Gen Genet (1987) 207:251 255 MGG © Springer-Verlag 1987 The Agrobacteriumrhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants Jens Stougaard, Dorte Abildsten, and Kjeld A. Marcker Department of Molecular Biology and Plant Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark Summary. A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. "Intermediate integration vectors" constructed from ColEl-derived plasmids served as cloning vectors in Escher- ichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agro- bacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. eorniculatus. Key words: Agrobacterium rhizogenes - Plant transforma- tion Intermediate integration vectors Binary vectors Introduction The neoplastic diseases crown gall and hairy root are caused by the soil bacterium Agrobacterium. The pathogenic prop- erties of the bacterium are encoded by two different large plasmids, tumour inducing (Ti) and root inducing (Ri), re- spectively. Both plasmids carry regions, T-DNA, which after transfer and expression from the plant genome cause the characteristic pathogenic traits (Hooykaas and Schilper- oort 1984, and references therein). T-DNA regions from Ti plasmids, named by their opine type, have been charac- terized genetically (Leemans et al. 1982; Joos et al. 1983; Willmitzer et al. 1982, 1983), and physically (Gielen et al. 1984; Barker et al. 1983). Genes involved in opine synthesis and synthesis of morphogenic phytohormones auxin and cytokinins were identified (Joos et al. 1983; Barry etal. 1984; Inz~ et al. 1984; Schr6der et al. 1981, 1984; Akiyoshi Offprint requests to: J. Stougaard et al. 1984). In the T-DNA only the 25 bp direct repeats bordering the T-DNA are required for the transfer of the DNA located in between (Zambryski et al. 1983; Wang et al. 1984), although transfer frequencies are increased by an adjacent short DNA sequence (Peralta et al. 1986). Ti- derived plant transformation vectors could therefore be constructed using the characterized T-DNA border se- quences (Zambryski et al. 1983; Bevan 1984). Like Ti plasmids Ri plasmids are characterised by their opine type (Petit et al. 1983). The agropine-type pRi has two T-DNA regions (De Paolis et al. 1985) called TL- and TR-DNA. The TL region is responsible for the hairy root phenotype and the TR for agropine synthesis as well as auxin biosynthesis. Four root morphogenic loci roIABCD are located in the TL segment and genes involved in auxin synthesis maps to the TR segment (White et al. 1985). Both the TL and TR segments are stably integrated into plant chromosomes and the 25 bp border repeats are found sur- rounding the TL segment (Constantino etal. 1984; Slightom et al. 1986). Presence of the TR is not required for the hairy root phenotype and root lines elicited by agro- pine-type A. rhizogenes strains do not always synthesize agropine (Petit et al. 1983; De Paolis et al. 1985). The A. rhizogenes transformation regeneration system available for a number of plant species (Chilton et al. 1982; David et al. 1984; Tepfer 1984; Petit et al. 1987) makes it desirable to construct pRi derived plant transformation vectors. We have therefore constructed a set of integration vectors and an A. rhizogenes strain carrying pBR322 se- quences within the TL-DNA of the agropine-type pRi plas- mid 15834. Also a binary vector was tested. These systems allow plant transformation without changing the hairy root inciting properties of the A. rhizogenes donor strains. Materials and methods Microbiological techniques. Escherichia coli and A. rhizo- genes strains were cultured in liquid LB medium or on LA plates, antibiotics were used at levels given by Van Haute et al. (1983). The plasmid helper system of Van Haute et al. (1983) was used for conjugational transfer of plasmids to A. rhizogenes. Plasmid constructions and DNA analysis. Standard DNA techniques as compiled by Maniatis et al. (1982) were used for DNA manipulations. Bacterial DNA for Southern blot