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Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia

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Summary

After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only TL-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both TL- and TR-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the TR-DNA markers were found. Not each of the TL-, or TR-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the TR-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.

From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the TR-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained TR-DNA. It also confirmed the absence of the TL-DNA, demonstrating the independent integration of the TR-region of the octopine-type Ti plasmid pTi Ach5.

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References

  1. Amasino RM, Powell ALT, Gordon MP: Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumour line. Mol Gen Genet 197:437–446, 1984.

    Google Scholar 

  2. Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350, 1983.

    Google Scholar 

  3. Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 6:1513–1523, 1979.

    Google Scholar 

  4. Caplan A, Herrera-Estrella L, Inzé D, VanHaute E, VanMontagu M, Schell J, Zambryski P: Introduction of genetic material into plant cells. Science 222:815–821, 1983.

    Google Scholar 

  5. Chang C, Chen C, Adams BR, Trost BM: Leucinopine, a characteristic compound of some crown-gall tumours. Proc Natl Acad Sci USA 80:3573–3576, 1983.

    Google Scholar 

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

    Google Scholar 

  7. Czakó M, Márton L. The efficiency of coculture transformation by Agrobacterium tumefaciens is influenced by growth regulators in Nicotiana. In preparation.

  8. Dahl GA, Guyon P, Petit A, Tempé J: Silver nitratepositive opines in corwn gall tumours. Plant Sci Lett 32:193–203, 1983.

    Google Scholar 

  9. DeBeuckeleer M, Lemmers M, DeVos G, Willmitzer L, VonMontagu M, Schell J: Further insight on the transferred DNA of octopine crown gall. Mol Gen Genet 183:283–288, 1981.

    Google Scholar 

  10. DeVos G, DeBeuckeleer M, VanMontagu M, Schell J: Restriction endonuclease mapping of the octopine tumour-inducing plasmid pTi Ach5 of Agrobacterium tumefaciens. Plasmid 6:249–253, 1981.

    Google Scholar 

  11. Ellis JG, Ryder MH, Tate ME: Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis. Mol Gen Genet 195:466–473, 1984.

    Google Scholar 

  12. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13, 1983.

    Google Scholar 

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

    Google Scholar 

  14. Gelvin SB, Karcher SJ, DiRita VJ: Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumours. Nucl Acids Res 11:159–173, 1983.

    Google Scholar 

  15. Gielen J, DeBeuckeleer M, Seurinck J, Deboeck F, DeGreve H, Lemmers M, VanMontagu M, Schell J: The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTi Ach5. EMBO J 3:835–846, 1984.

    Google Scholar 

  16. Guyon P, Chilton MD, Petit A, Tempé J: Agropine in ‘null type’ tumours: evidence for generality of the opine concept. Proc Natl Acad Sci USA 77:2693–2697, 1980.

    Google Scholar 

  17. Hooykaas PJJ, Schilperoort RA: The molecular genetics of crown gall tumorigenesis. Adv Gen 22:209–283, 1984.

    Google Scholar 

  18. Klapwijk PM, VanBreukelen J, Korevaar K, Ooms G, Schilperoort RA. Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens. J Bacteriol 141:129–136, 1980.

    Google Scholar 

  19. Leemans J, Shaw C, DeBlaere R, DeGreve H, Hernalsteens JP, Maes M, VanMontagu M, Schell J: Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet 1:149–164, 1981.

    Google Scholar 

  20. Leemans J, Deblaere R, DeGreve H, Hernalsteens JP, VanMontagu M, Schell J: Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152, 1982.

    Google Scholar 

  21. Leemans J, Hernalsteens JP, Deblaere R, DeGreve H, Thia-Toong H, VanMontagu M, Schell J: Genetic analysis of T-DNA and regeneration of transformed plants: In: Pühler A (ed) Molecular Genetics of the Bacteria-Plant Interaction. Springer-Verlag, Berlin/Heidelberg, 1983, pp 322–330.

    Google Scholar 

  22. Lemmers M, DeBeuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens JP, VanMontagu M, Schell J: Internal organization, boundaries and integration of Ti plasmid DNA in nopaline crown gall tumours. J Mol Biol 144:353–376, 1980.

    Google Scholar 

  23. Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982, pp 224–228.

    Google Scholar 

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

    Google Scholar 

  25. Márton L: Transformation of tobacco cells by coculture with Agrobacterium tumefaciens. In: Vasil I (ed) Somatic Cell Genetics of Plants, Vol. 1. Academic Press, New York, 1984, pp 514–521.

    Google Scholar 

  26. Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:472–497, 1962.

    Google Scholar 

  27. Nester EW, Gordon MP, Amasino RM, Yanofsky MF: Crown gall: a molecular and physiological analysis. Ann Rev Plant Physiol 35:387–413, 1984.

    Google Scholar 

  28. Ooms G, Hooykaas PJJ, Molenaar G, Schilperoort RA: Crown gall plant tumours of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti-plasmids; Analysis of T-DNA functions. Gene 14:33–50, 1981.

    Google Scholar 

  29. Ooms G, Bakker A, Molendijk L, Wullems GJ, Gordon MP, Nester EW, Schilperoort RA: T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum. Cell 30:589–597, 1982.

    Google Scholar 

  30. Otten LABM, Schilperoort RA: A rapid microscale method for the detection of lysopine and nopaline dehydrogenase. Biochim Biophys Acta 527:497–500, 1978.

    Google Scholar 

  31. Radloff R, Bauer W, Vinograd J: A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in Hela cells. Proc Natl Acad Sci USA 57:1514–1521, 1967.

    Google Scholar 

  32. Salomon F, Deblaere R, Leemans J, Hernalsteens JP, VanMontagu M, Schell J: Genetic identification of functions of TR-DNA transcripts in octopine crown galls. EMBO J 3:141–146, 1984.

    Google Scholar 

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

    Google Scholar 

  34. Sidorov VA, Maliga P: Fusion-complementation analysis of auxotrophic and chlorophyll-deficient lines isolated in haploid Nicotiana plumbaginifolia protoplast cultures. Mol Gen Genet 186:328–332, 1982.

    Google Scholar 

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

    Google Scholar 

  36. Tate ME, Ellis JG, Kerr A, Tempé J, Murray KE, Shaw KJ: Agropine: a revised structure. Carbohydr Res 104:105–120, 1982.

    Google Scholar 

  37. Taylor B, Powell A: Isolation of plant DNA and RNA. Focus 4:3–4, 1983.

    Google Scholar 

  38. Tempé J, Guyon P, Petit A, Ellis JC, Tate ME, Kerr A: Préparation et propriétés de nouveaux substrats cataboliques pour deux types de plasmides oncogénes d' Agrobacterium tumefaciens: CR Hebd Seances Acad Sci, Série D 290:1173–1176, 1980.

    Google Scholar 

  39. Tempé J, Petit A: La Piste des Opines. In: Pühler A (ed) Molecular Genetics of the Bacteria-Plant Interaction. Springer-Verlag, Berlin/Heidelberg, 1983, pp 13–32.

    Google Scholar 

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

    Google Scholar 

  41. Trevelyan WE, Procter DP, Harrison JS: Detection of sugars on paper chromatograms. Nature (London) 166:444–446, 1980.

    Google Scholar 

  42. VanSlogteren GMS, Hooykaas PJJ, Schilperoort RA: Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment. Plant Mol Biol 3:333–336, 1984.

    Google Scholar 

  43. Velten J, Willmitzer L, Leemans J, Ellis J, Deblaere R, VanMontagu M, Schell J: TR genes involved in agropine production. In: Pühler A (ed) Molecular Genetics of the Bacteria-Plant Interaction. Springer-Verlag, Berlin/Heidelberg, 1983, pp 303–312.

    Google Scholar 

  44. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683–3687, 1979.

    Google Scholar 

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

    Google Scholar 

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  1. Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701, Szeged, Hungary

    Mihály Czakó & László Márton

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  1. Mihály Czakó
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  2. László Márton
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Czakó, M., Márton, L. Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia . Plant Mol Biol 6, 101–109 (1986). https://doi.org/10.1007/BF00027303

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  • DOI: https://doi.org/10.1007/BF00027303

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