Journal of General Microbiology (1977), 102, 427-430. Printed in Great Britain 427 Variations in Plasmid DNA Sequences Present in Crown Gall Tumour Lines ByANN G. MATTHYSSE Department of Botany, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A. (Received 27 May 1977) INTRODUCTION Extracellular infections of wounded dicotyledonous plants by the Gram-negative bacterium Agrobmterium tumefaciens (Smith & Townsend) Conn result in the transformation of normal plant cells to tumour cells. These crown gall tumour cells will continue to grow autonomously in the absence of the bacterium (Braun, 1943). Virulent strains of A. tume- faciens contain a large plasmid (120 x 1oS molecular weight; Zaenen et al., 1974). The pres- ence of this plasmid is correlated with the virulence of the bacterium (Van Larebeke et al., 1974). Bacteria which have lost the plasmid are avirulent and regain virulence when the plasmid is reintroduced into them (Watson et al., 1975). One strain of crown gall tumour cells, Vinca rosea v,,, has been shown to contain DNA sequences complementary to more than of the bacterial plasmid DNA (Matthysse & Stump, 1976). In order to determine whether this tumour strain is unique, two other bacteria-free strains of crown gall tumours have been examined for the presence of DNA sequences complementary to plasmid DNA. METHODS Media. Agrobacterium tumefaciens strains BP and ~6 were grown in minimal salts medium containing (g 1-'): NH4CI, 5; NH,N03, I; Na,SO,, 2; K2HP04, 3; KH2P04, I; MgS04.7H20, 0.1; with 0.2% (w/v) glucose and 50 to roo pCi [met/~yl-~H]thymidine m1-l (Clowes & Hayes, 1968). Vinca rosea crown gall tumour tissue culture cells were grown on unsupplemented White's medium (Wood & Braun. 1961). Normal V. rosea tissue culture cells were grown on White's medium supplemented with (mg I-'): a-naphthalene acetic acid (NAA), I ; kinetin, 0.5; asparagine, 200; glutamine, 200; meso-inositol. 100; AMP, 100; GMP, 100 (Wood & Braun, 1961). Normal tobacco was grown on Murashige & Skoog's (1962) medium supple- mented with 5 mg NAA 1-' and I mg kinetin 1-'. Bryophyllum tissue cultures were grown on Miller's medium (1961) containing I g citric acid I-' and 2 mg NAA I-l. Organisms. Agrobacterium tumefaciens strain BP and V. rosea normal strain NV and crown gall tissue culture strain vBP were obtained from Professor A. Braun. Tissue cultures of normal Nicotiana tabacum were obtained from P. Wyman. Agrobacterium turnefaciens strain ~6 and Bryophyllurn crown gall tissue culture strain BB~ were obtained from Professor C. Miller. Isolation of DNA. Plant cell DNA was extracted by the method of Kirby (1957) as previously described (Matthysse & Stump, 1976). Purified DNA preparations in 0.1 x SSC (saline sodium citrate, where I x SSC contains 0.15 M-NaCI and 0.015 M-trisodium citrate) had E260/E280 ratios between 1.75 and 1.95. No RNA was detectable by examination of the supernatant from the base hydrolysis (0.3 M-KOH, 18 h, 37 "C) of 100pg DNA ml-' for E280. Bacterial plasmid DNA was prepared as previously described (Matthysse & Stump, 1976) or by CsCl- ethidium bromide equilibrium density gradient centrifugation as described by Klein et al. (1975). Each of the strains used appears, from restriction endonuclease digestion patterns, to contain only one type of plasmid (Mandelkorn & Matthysse, unpublished observation). The specific activity of the tritiated bacterial plasmid DNA ranged from 2 x 105 to I x 10' c.p.m. pg-'. Before use in hybridization reactions, both the plant and plasmid DNAs were degraded to a molecular weight of approximately 2 x IO~ by a limited depurination reaction (McConaughy & McCarthy, 1967). The DNA was dissoIved in 0.1 M-sodium acetate pH 4.2 and heated at 70 "C for 33 min. The pH of the solution was then adjusted to I 2 with NaOH and the solution was heated at 50 "C for 10 min. It was then dialysed against 1-5 x SSC.