Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Summary

With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T+), also had acquired non-selected calf thymus carrier DNA sequences (C+), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas+), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (‘transforming’) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C+T+. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C+T+:C-T+: C+T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.

This is a preview of subscription content, access via your institution.

References

  1. 1.

    Akiyoshi DE, Klee H, Amasino RM, Nester EW, Gordon MP: T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci USA 81: 5994–5998, 1984.

    Google Scholar 

  2. 2.

    Anderson RA, Krakauer T, Camerini-Otero RD: DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid. Proc Natl Acad Sci USA 79: 2748–2752, 1982.

    Google Scholar 

  3. 3.

    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 

  4. 4.

    Barry GF, Rogers SG, Fraley RT, Brand L: Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 81: 4776–4780, 1984.

    Google Scholar 

  5. 5.

    Bevan MW, Flavell RB, Chilton M-D: A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304: 184–187, 1983.

    Google Scholar 

  6. 6.

    Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711–8721, 1984.

    Google Scholar 

  7. 7.

    Block Mde, Herrera-Estrella L, Montagu Mvan, Schell J, Zambryski P: Expression of foreign genes in regenerated plants and in their progeny. EMBO J 3: 1681–1689, 1984.

    Google Scholar 

  8. 8.

    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 

  9. 9.

    Herrera-Estrella L, Broeck Gvan den, Maenhout R, Montagu Mvan, Schell J, Timko M, Cashmore A: Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature 310: 115–120, 1984.

    Google Scholar 

  10. 10.

    Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180, 1983a.

    Google Scholar 

  11. 11.

    Hoekema A, Haaren MJJvan, Hille J, Hoge JHC, Hooykaas PJJ, Krens FA, Wullems GJ, Schilperoort RA: Agrobacterium tumefaciens and its Ti-plasmid as tools in transformation of plant cells. In: Goldberg R (ed) Plant molecular biology. Alan R Liss, New York, 1983b, pp 3–22.

    Google Scholar 

  12. 12.

    Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A, Hoffmann N: Inheritance of functional foreign genes in plants. Science 223: 496–498, 1984.

    Google Scholar 

  13. 13.

    Krens FA, Molendijk L, Wullems GJ, Schilperoort RA: In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72–74, 1982.

    Google Scholar 

  14. 14.

    Krens FA, Schilperoort RA: T-DNA uptake and expression by protoplasts of Nicotiana tabacum. In: Vasil IK (ed) Cell culture and somatic cell genetics of plants. Academic Press, New York, 1984, pp 522–534.

    Google Scholar 

  15. 15.

    Krens FA, Mans RMW, Slogteren TMS, Hoge JHC, Wullems GJ, Schilperoort RA: Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences. Plant Mol Biol 5: 223–234, 1985.

    Google Scholar 

  16. 16.

    Leemans J, Deblaere R, Willmitzer L, Greve Hde, Hernalsteens J-P, Montagu Mvan, Schell J: Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1: 147–152, 1982.

    Google Scholar 

  17. 17.

    Linsmaier EM, Skoog F: Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 8: 100–127, 1965.

    Google Scholar 

  18. 18.

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

    Google Scholar 

  19. 19.

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

    Google Scholar 

  20. 20.

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

    Google Scholar 

  21. 21.

    Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722, 1984.

    Google Scholar 

  22. 22.

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

    Google Scholar 

  23. 23.

    Scangos G, Ruddle FH: Mechanisms and applications of DNA-mediated gene transfer in mammalian cells a review. Gene 14: 1–10, 1981.

    Google Scholar 

  24. 24.

    Schaefer A, Ohyama K, Gamborg OL: Detection by agarose gel electrophoresis of nucleases associated with cells and protoplasts from plant suspension cultures using Agrobacterium tumefaciens Ti plasmid Agric Biol Chem 45: 1441–1445, 1981.

    Google Scholar 

  25. 25.

    Schroeder G, Waffenschmidt S, Weiler EW, Schroeder J: The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem 138: 387–391, 1984.

    Google Scholar 

  26. 26.

    Simpson RB, O'Hara PJ, Kwok W, Montoya AL, Lichtenstein C, Gordon MP, Nester EW: DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA. Cell 29: 1005–1014, 1982.

    Google Scholar 

  27. 27.

    Slogteren GMS, Hoge JHC, Hooykaas PJJ, Schilperoort RA: Clonal analysis of heterogeneous crown gall tumor tissues induced by wild-type and shooter mutant strains of Agrobacterium tumefaciens-expression of T-DNA genes. Plant Mol Biol 2: 321–333, 1983.

    Google Scholar 

  28. 28.

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

    Google Scholar 

  29. 29.

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

    Google Scholar 

  30. 30.

    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 

  31. 31.

    Yadav NS, Vanderleyden J, Bennett DR, Barnes WM, Chilton M-D: Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci USA 79: 6322–6326, 1982.

    Google Scholar 

  32. 32.

    Zambryski P, Depicker A, Kruger K, Goodman HM: Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA J Mol Appl Genet 1: 361–370, 1982.

    Google Scholar 

  33. 33.

    Zambryski P, Joos H, Genetello C, Leemans J, Montagu Mvan, Schell J: Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J 2: 2143–2150, 1983.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Peerbolte, R., Krens, F.A., Mans, R.M.W. et al. Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences. Plant Mol Biol 5, 235–246 (1985). https://doi.org/10.1007/BF00020641

Download citation

Keywords

  • cotransformation
  • DNA-transformation
  • N. tabacum
  • progeny
  • Ti plasmid