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Sexual transmission of T-DNA in abnormal tobacco regenerants transformed by octopine and nopaline strains of Agrobacterium tumefaciens

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Summary

Transformed shoots regenerating from Crown gall tissues were produced by in vitro infection of regenerating tobacco protoplasts with Agrobacterium tumefaciens. As these shoots were abnormal in their growth and were unable to form roots, they were grafted onto normal tobacco, resulting in the formation of flowering shoots. Sexual crosses between different grafts and selfing produced two different types of seedlings. Some developed into normal, untransformed plants whereas others failed to develop a root system and remained very small. The abnormal seedlings were shown to consist of transformed cells since they expressed the opine marker. The suppression of root formation and the production of opines were 100 percent linked. The analysis of the progeny indicated that the shoots derived from these tumour lines were hemizygous for the T-DNA linked genes and expressed these genes as a dominant locus. The opine-positive shoots were shown to contain a deleted T-DNA and also some T-DNA specific transcripts: the transcript for octopine synthase or LpDH and two other transcripts, one of which is known to be involved in the suppression of root formation. The same transcripts could also be detected in the sexual progeny. These observations confirm that some active tumour-controlling genes can be sexually transmitted and do not drastically interfere with seed development, germination and early shoot formation. The formation of roots by these seedlings is however severely suppressed.

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Abbreviations

LpDH:

Lysopine Dehydrogenase

NpDH:

Nopaline Dehydrogenase

References

  • Akiyoshi DE, Morris RO, Hinz R, Mischke BS, Kosuge T, Garfinkel DJ, Gordon MP, Nester EW (1983) Cytokinin-auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80:407–411

    Google Scholar 

  • Amasino RM, Miller CO (1983) Effect of temperature on the morphology and cytokinin levels of tobacco crown gall teratoma tissues. Plant Sci Lett 28:245–253

    Google Scholar 

  • Barton KA, Binns AN, Matzke AJM, Chilton M-D (1983) Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA and transmission of T-DNA to R1 progeny. Cell 32:1033–1043

    Google Scholar 

  • Bedbrook J (1981) A plant nuclear DNA preparation procedure. Plant Mol Biol Newslett, vol II, 24

    Google Scholar 

  • Binns AN, Sciaky D, Wood HW (1982) Variation in hormone autonomy and regenerative potential of cells transformed by strain A66 of Agrobacterium tumefaciens. Cell 31:605–612

    Google Scholar 

  • Braun AC(1959) A demonstration of the recovery of the crown-gall tumor cell with the use of complex tumors of single-cell origin. Proc Natl Acad Sci USA 45:932–938

    Google Scholar 

  • Braun AC, White PR (1943) Bacteriological sterility of tissues derived from secondary crown gall tumors. Phytopathol 33:85–100

    Google Scholar 

  • Braun AC, Wood HN (1976) Suppression of the neoplastic state with the acquisition of specialized functions in cells, tissues, and organs of crown gall teratomas of tobacco. Proc Natl Acad Sci USA 73:496–500

    Google Scholar 

  • Chilton M-D, Drummond MH, Merlo DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–271

    Google Scholar 

  • Chilton M-D, Saiki RK, Yadav N, Gordon MP, Quetier F (1980) T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci USA 77:4060–4064

    Google Scholar 

  • De Beuckeleer M, Lemmers M, De Vos, G, Willmitzer L, Van Montagu M, Schell J (1981) Further insight on the transferred-DNA of octopine crown gall. Mol Gen Genet 183:283–288

    Google Scholar 

  • De Greve H, Leemans J, Hernalsteens J-P, Thia-Toong L, De Beuckeleer M, Willmitzer L, Otten L, Van Montagu M, Schell J (1982) Regeneration of normal and fertile plants that express octopine synthase, from tobacco crown galls after deletion of tumor-controlling functions. Nature (Lond) 300:752–755

    Google Scholar 

  • De Greve H, Dhaese P, Seurinck J, Lemmers M, Van Montagu M, Schell J (1983) Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1:499–511

    Google Scholar 

  • Denhard DT (1966) A membranefilter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23:641–646

    Google Scholar 

  • De Vos G, De Beuckeleer M, Van Montagu M, Schell J (1981) Restriction endonuclease mapping of the octopine tumor inducing pTiAch5 of Agrobacterium tumefaciens. Plasmid 6:249–253

    Google Scholar 

  • Drummond MH, Gordon MP, Nester EW, Chilton M-D (1977) Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumors. Nature (Lond) 269:535–536

    Google Scholar 

  • Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW (1981) Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153

    Google Scholar 

  • Gelvin SB, Gordon MP, Nester EW, Aronson AI (1981) Transcription of the Agrobacterium Ti-plasmid in the bacterium and in crown gall tumors. Plasmid 6:17–29

    Google Scholar 

  • Gurley WB, Kemp JD, Albert MJ, Sutton DW, Callis J (1979) Transcription of Ti plasmid derived sequences in three octopine-type crown gall tumor lines. Proc Natl Acad Sci USA 76:2828–2832

    Google Scholar 

  • Hernalsteens J-P, Van Vliet F, De Beuckeleer M, Depicker A, Engler G, Lemmers M, Holsters M, Van Montagu M, Schell J (1980) The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. Nature (Lond) 287:654–656

    Google Scholar 

  • Joos H, Inze D, Caplan A, Sormann M, Van Montagu M, Schell J (1983) Genetic analysis of T-DNA transcripts in nopaline crown gall. Cell 32:1057–1067

    Google Scholar 

  • Klapwijk PM, Van Breukelen J, Korevaar K, Ooms G, Schilperoort RA (1980) Transposition of Tn904 encoding Sm resistance into the octopine Ti-plasmid of A. tumefaciens. J Bacteriol 141:129–136

    Google Scholar 

  • Leemans J, Shaw C, Deblaere R, De Greve H, Hernalsteens J-P, Maes M, Van Montagu M, Schell J (1981) Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet 1:149–164

    Google Scholar 

  • Leemans J, Deblaere R, Willmitzer L, De Greve H, Hernalsteens J-P, Van Montagu M, Schell J (1982) Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152

    Google Scholar 

  • Lemmers M, De Beuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens J-P, Van Montagu M, Schell J (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumors. J Mol Biol 144:353–376

    Google Scholar 

  • Maliga P, Sz Breznovits A, Márton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nature (Lond) 244:29–30

    Google Scholar 

  • Márton L, Wullems GJ, Molendijk L, Schilperoort RA (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature (Lond) 277:129–131

    Google Scholar 

  • Memelink J, Wullems GJ, Schilperoort RA (1983) Nopaline T-DNA is maintained during regeneration and generative propagation of transformed tobacco plants. Mol Gen Genet 190:516–522

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Google Scholar 

  • Ooms G, Hooykaas PJJ, Moolenaar G, Schilperoort RA (1981) Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti-plasmid; analysis of T-DNA functions. Gene 14:33–50

    Google Scholar 

  • Otten LABM, Schilperoort RA (1978) A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500

    Google Scholar 

  • Otten L, De Greve H, Hernalsteens J-P, Van Montagu M, Schieder O, Straub J, Schell J (1981) Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Mol Gen Genet 183:209–213

    Google Scholar 

  • Schell J, Van Montagu M (1977) The Ti plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of NIF genes in plants? In: Hollaender A (ed) Genetic engineering for nitrogen fixation. Plenum Press, New York, pp 159–179

    Google Scholar 

  • Schell J, Van Montagu M, De Beuckeleer M, De Block M, Depicker A, De Wilde M, Engler G, Genetello C, Hernalsteens J-P, Holsters M, Seurinck J, Silva B, Van Vliet F, Villarroel R (1979) Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proc R Soc Lond B 204:251–266

    Google Scholar 

  • Schilperoort RA, Klapwijk PM, Hooykaas PJJ, Koekman BP, Ooms G, Otten LABM, Wurzer-Figurelli EM, Wullems GJ, Rörsch A (1978) A. tumefaciens plasmids as vectors for genetic transformation of plant cells. In: Thorpe TA (ed) Frontiers of plant tissue culture 1978. The International Association for Plant Tissue Culture 1978. University of Calgary, Alberta, Canada, pp 85–94

    Google Scholar 

  • Schröder J, Schröder G, Huisman H, Schilperoort RA, Schell J (1981) The mRNA for lysopine dehydrogenase in plant tumor cells is complementary to a Ti-plasmid fragment. FEBS Lett 129:166–168

    Google Scholar 

  • Smith EF, Townsend CO (1907) A plant tumor of bacterial origin. Science 25:671–673

    Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Google Scholar 

  • Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW (1980a) Integration and organization of Ti-plasmid sequences in crown gall tumors. Cell 19:729–739

    Google Scholar 

  • Thomashow MF, Nutter R, Postle K, Chilton M-D, Blattner FR, Powell A, Gordon MP, Nester EW (1980b) Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens. Proc Natl Acad Sci USA 77:6448–6452

    Google Scholar 

  • Trevelyan W, Proctor D, Harrison J (1950) Detection of sugars on paper chromatograms. Nature 166:444–445

    Google Scholar 

  • Turgeon R, Wood HN, Braun AC (1976) Studies on the recovery of crown gall tumor cells. Proc Natl Acad Sci USA 73:3562–3564

    Google Scholar 

  • Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature (Lond) 252:169–170

    Google Scholar 

  • Watson B, Currier TC, Gordon MP, Chilton M-D, Nester EW (1975) plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol 123:255–264

    Google Scholar 

  • Willmitzer L, De Beuckeleer M, Lemmers M, Van Montagu M, Schell J (1980) The Ti-plasmid derived T-DNA is present in the nucleus and absent from plastids of plant crown-gall cells. Nature (Lond) 287:359–361

    Google Scholar 

  • Willmitzer L, Otten L, Simons G, Schmalenbach W, Schröder J, Schröder G, Van Montagu M, De Vos G, Schell J (1981a) Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures. Mol Gen Genet 182:255–262

    Google Scholar 

  • Willmitzer L, Schmalenbach W, Schell J (1981b) Transcription of T-DNA in octopine and nopaline crown gall tumors is inhibited by low concentrations of α-amanitin. Nucl Acids Res 9:4801–4812

    Google Scholar 

  • Willmitzer L, Simons G, Schell J (1982) The TL-DNA in octopine crown gall tumors codes for seven well defined polyadenylated transcripts. EMBO J 1:139–146

    Google Scholar 

  • Willmitzer L, Dhaese P, Schreier PH, Schmalenbach W, Van Montagu M, Schell J (1983) Size, location and polarity of T-DNA encoded transcripts in nopaline crown gall tumors; common transcripts in octopine and nopaline tumors. Cell 32:1045–1056

    Google Scholar 

  • Wullems GJ, Molendijk L, Ooms G, Schilperoort RA (1981) Rentention of tumor markers in F1 progeny plants from in vitro induced octopine and nopaline tumor tissues. Cell 24:719–727

    Google Scholar 

  • Yadav NS, Postle K, Saiki RK, Thomashow MF, Chilton M-D (1980) T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature (Lond) 287:458–461

    Google Scholar 

  • Yang F, Simpson RB (1981) Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences. Proc Natl Acad Sci USA 78:4151–4155

    Google Scholar 

  • Zaenen I, Van Larebeke N, Teuchy H, Van Montagu M, Schell J (1974) Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J Mol Biol 86:109–127

    Google Scholar 

  • Zambryski P, Holsters M, Kruger K, Depicker A, Schell J, Van Montagu M, Goodman HM (1980) Tumor DNA structure in plant cells transformed by A. tumefaciens. Science 209:1385–1391

    Google Scholar 

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Authors and Affiliations

  1. Abt. Schell, Max Planck Institut für Züchtungsforschung, D-5000, Köln30 (Vogelsang), Germany

    Anke Wöstemeyer, Léon A. B. M. Otten & Jozef St. Schell

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  1. Anke Wöstemeyer
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  2. Léon A. B. M. Otten
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  3. Jozef St. Schell
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Communicated by R. Goldberg

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Wöstemeyer, A., Otten, L.A.B.M. & Schell, J.S. Sexual transmission of T-DNA in abnormal tobacco regenerants transformed by octopine and nopaline strains of Agrobacterium tumefaciens . Molec. Gen. Genet. 194, 500–507 (1984). https://doi.org/10.1007/BF00425565

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  • DOI: https://doi.org/10.1007/BF00425565

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