Abstract
An approach is described for the detection of a unique sequence, the T-DNA region of the Agrobacterium rhizogenes root-inducing (Ri) plasmid, in plant chromosomes by in situ hybridization. This sequence was introduced into the Crepis capillaris genome (2n=6) by infecting Crepis stem segments with A. rhizogenes. Roots growing from the infection site contain T-DNA and synthesize mannopine, which can be used as a convenient biochemical marker for T-DNA transformation. Southern analysis of DNA isolated from one transformed Crepis root line verified the presence of a single copy of T-DNA (approximate size 17 kb) per diploid Crepis genome. To localize T-DNA, both DNA and RNA probes, labelled with either tritium or biotin, were hybridized to Crepis chromosomes prepared from transformed root tips by a novel spreading method. Biotinylated probes were visualized using reflection-contrast microscopy. In the hybridization experiments described, T-DNA was detected in one homologue of chromosome 3, where it could be assigned to a paracentromeric position in the neighbourhood of the nucleolar organizing region. These results demonstrate that it is possible to localize unique sequences in plant chromosomes by in situ hybridization.
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Ashmore SE, Gould AR (1981) Karyotype evolution in a tumor derived plant tissue culture analyzed by giemsa C-banding. Protoplasma 106:297–308
Bennett MD, Smith JB, Heslop-Harrison JS (1982) Nuclear DNA amounts in angiosperms. Proc R Soc Lond B 216:179–199
Bevan MW, Chilton M-D (1982) T-DNA of the Agrobacterium Ti and Ri plasmid. Annu Rev Genet 16:357–384
Byrne MC, Koplow J, David C, Tempé J, Chilton M-D (1983) Structure of T-DNA in roots transformed by Agrobacterium rhizogenes. J Mol Appl Gen 2:201–209
Chilton M-D, Tepfer D, Petit A, David C, Casse-Delbart F, Tempé J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995
Cox K, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485–502
Dahl G, Guyon P, Petit A, Tempé J (1983) Silver nitrate-positive opines in crown gall tumors. Plant Sci Lett 32:193–203
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21
Gall J, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63:378–383
Henderson AS (1982) Cytological hybridization to mammalian chromosomes. Int Rev Cytol 76:1–46
Hennig W (1973) Molecular hybridization of DNA and RNA in situ. Int Rev Cytol 36:1–41
Jhanwar SC, Neel BG, Hayward WS, Chaganti RSK (1983) Localization of c-ras oncogene family on human germ line chromosomes. Proc Natl Acad Sci USA 80:4794–4797
Koplow J, Byrne MC, Jen G, Tempé J, Chilton M-D (1984) Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid. Plasmid 11:17–27
Landegent JE, Jansen in de Wal N, Ploem JS, van der Ploeg M (1985a) Sensitive detection of hybridocytochemical results by means of reflection-contrast microscopy. J Histochem Cytochem 33:1241–1246
Landegent JE, Jansen in de Wal N, van Ommen GJ-B, Baas F, de Vijlder JJM, van Duijn P, van der Ploeg M (1985b) Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317:175–177
Manuelidis L, Langer-Safer PR, Ward DC (1982) High-resolution mapping of satellite DNA using biotin-labelled DNA probes. J Cell Biol 95:619–625
Matzke AJM, Chilton M-D (1981) Site-specific insertion of genes into T-DNA of the Agrobacterium tumefaciens Ti plasmid: an approach to genetic engineering of higher plant cells. J Mol Appl Genet 1:39–49
Murashige T, Skoog F (1962) A revised medium for growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Rapp AK, Marijnen JGJ, Vrolijk J, van der Ploeg M (1986) Denaturation, renaturation and loss of DNA during in situ hybridization procedures. Cytometry, in press
Rayburn AL, Gill BS (1985) Use of biotin-labeled probes to map specific DNA sequences on wheat chromosomes. J Hered 76:78–81
Robins DM, Ripley S, Henderson AS, Axel R (1981) Transforming DNA integrates into the host chromosome. Cell 23:29–39
Sacristán MD, Wendt-Gallitelli MF (1973) Tumorous cultures of Crepis capillaris: chromosomes and growth. Chromosoma 43:279–288
Schweizer D (1973) Differential staining of plant chromosomes with giemsa. Chromosoma 40:307–320
Szabo P, Ward DC (1982) What's new with hybridization in situ. Trends Biochem Sci 7:425–427
Tanaka R, Komatsu H (1977) C-banding patterns in somatic and meiotic chromsomes of Crepis capillaris. Proc Jpn Acad B 53(I):6–9
Thomashow MF, Nutter R, Montoya A, Gordon M, Nester E (1980) Integration and organization of Ti plasmid sequences in crown gall tumors. Cell 19:729–735
Verschueren H (1985) Interference reflection microscopy in cell biology: methodology and applications. J Cell Sci 75:279–301
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Ambros, P.F., Matzke, M.A. & Matzke, A.J.M. Detection of a 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94, 11–18 (1986). https://doi.org/10.1007/BF00293525
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DOI: https://doi.org/10.1007/BF00293525