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Efficient Transformation of Alfalfa Protoplasts by the Intranuclear Microinjection of Ti Plasmids

Bio/Technology volume 4, pages 1001–1004 (1986)Cite this article

Abstract

Intranuclear microinjection of alfalfa (Medicago sativa L.) protoplasts yielded transformation frequencies of 15–26%. Over 70 transformed callus lines were recovered without selection by microinjecting a variety of plasmids. Analyses of several lines transformed with pTiC58 showed that integration did not occur by the T–DNA mechanism typical for crown gall tissues. The presence of a functional T–DNA right border on smaller plasmids or the coinjection of a functional vir region on a separate plasmid did not increase the transformation frequencies. The novelty of this approach to the genetic transformation of plants is that selection systems are not required and restrictions on the host range of Agrobacterium tumefaciens may be circumvented.

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References

  1. Hurkman, W.J., Smith, L.D., Richter, J., and Larkins, B.A. 1981. Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs. J. Cell Biol. 89:292–299.

    Article  CAS  Google Scholar 

  2. Palevitz, B.A., and Hepler, P.K. 1985. Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer Yellow. Planta 164:473–479.

    Article  CAS  Google Scholar 

  3. Matthews, J.A., Brown, J.W.S., and Hall, T.C. 1981. Phaseolin mRNA is translated to yield glycosylated polypeptides in Xenopus oocytes. Nature 294:175–176.

    Article  CAS  Google Scholar 

  4. Wagner, T.E., Hoppe, P.C., Jollick, J.D., Scholl, D.R., Hodinka, R.L., and Gault, J.B. 1981. Microinjection of a rabbit β-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. USA 78:6376–6380.

    Article  CAS  Google Scholar 

  5. Palmiter, R.D., Chen, H.Y., and Brinster, R.L. 1982. Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell 29:701–710.

    Article  CAS  Google Scholar 

  6. Rubin, G.M., and Spradling, A.C. 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353.

    Article  CAS  Google Scholar 

  7. Steinbiss, H.-H., and Stabel, P. 1983. Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow. Protoplasma 116:223–227.

    Article  Google Scholar 

  8. Lawrence, W.A., and Davies, D.R. 1985. A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep. 4:33–35.

    Article  CAS  Google Scholar 

  9. Morikawa, H., and Yamada, Y. 1985. Capillary microinjection into protoplasts and intranuclear localization of injected materials. Plant Cell Physiol. 26:229–236.

    CAS  Google Scholar 

  10. Crossway, A., Oakes, J.V., Irvine, J.M., Ward, B., Knauf, V.C., and Shewmaker, L.K. 1986. Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Molec. Gen. Genet. 202:179–185.

    Article  CAS  Google Scholar 

  11. Reich, T.J., Iyer, V.N., Scobie, B., and Miki, B.L. 1986. A detailed procedure for the intranuclear microinjection of plant protoplasts. Can. J. Bot. In press.

  12. Reich, T.J., Iyer, V.N., Haffner, M., Holbrook, L.A., and Miki, B.L. 1986. The use of fluorescent dyes in the microinjection of alfalfa protoplasts. Can. J. Bot. In press.

  13. Griesbach, R.J. 1983. Protoplast microinjection. Plant Mol. Biol. Rep. 1:32–37.

    Article  Google Scholar 

  14. Holbrook, L.A., Reich, T.J., Iyer, V.N., Haffner, M., and Miki, B.L. 1985. Induction of efficient cell division in alfalfa protoplasts. Plant Cell Rep. 4:229–232.

    Article  CAS  Google Scholar 

  15. Meadows, M.G., and Potrykus, I. 1981. Hoechst 33258 as a vital stain for plant cell protoplasts. Plant Cell Rep. 1:77–79.

    Article  CAS  Google Scholar 

  16. Horsch, R.B., and Jones, G.E. 1980. A double filter paper technique for plating cultured plant cells. In Vitro 16:103–108.

    Article  Google Scholar 

  17. Otten, L.A.B.M., and Schilperoort, R.A. 1978. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim. Biophys. Acta 527:497–500.

    Article  CAS  Google Scholar 

  18. Aerts, M., Jacobs, M., Hernalsteens, J.-P., Van Montagu, M., and Schell, J. 1979. Induction and in vitro culture of Arabidopsis thaliana crown gall tumours. Plant Sci. Lett. 17:43–50.

    Article  CAS  Google Scholar 

  19. Holsters, M., Silva, B., Van Vliet, F. et al. 1980. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230.

    Article  CAS  Google Scholar 

  20. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman, H.M. 1982. Nopalinc synthase: Transcript mapping and DNA sequence. J. Mol. Appl. Genet. 1:561–573.

    CAS  Google Scholar 

  21. Holsters, M., Villaroel, R., Van Montagu, M., and Schell, J. 1982. The use of selectable markers for the isolation of plant-DNA/T-DNA junction fragments in a cosmid vector. Mol. Gen. Genet. 185:283–289.

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  23. Wang, K., Herrera-Estrella, L., Van Montagu, M., and Zambryski, P. 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455–462.

    Article  CAS  Google Scholar 

  24. Depicker, A., De Wilde, M., De Vos, G., De Vos, R., Van Montagu, M., and Schell, J. 1980. Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid 3:193–211.

    Article  CAS  Google Scholar 

  25. Fraley, R.T., Rogers, S.G., Horsch, R.B., Eicholtz, J.S., Fink, C.L., Hoffmann, N.L., and Sanders, P.R. 1985. The SEV system: A new disarmed Ti plasmid vector system for plant transformation. Bio/Technology 3:629–635.

    CAS  Google Scholar 

  26. Fraley, R.T., Rogers, S.G., Horsch, R.B. et al. 1983. Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80:4803–4807.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Nims, R.C., Halliwell, R.S., and Rosberg, D.W. 1967. Wound healing in cultured tobacco cells following microinjection. Protoplasma 64:305–314.

    Article  Google Scholar 

  29. Capecchi, M.R. 1980. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22:479–488.

    Article  CAS  Google Scholar 

  30. Brinster, R.L., Chen, H.Y., Trumbauer, M., Yagle, M.K., and Palmiter, R.D. 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82:4438–4442.

    Article  CAS  Google Scholar 

  31. Krens, F.A., Mans, R.M., van Slogteren, J.M.S., Hoge, J.H.C., Wullems, G.J., and Schilperoort, R.A. 1985. Structure and expression of DNA transformed to tobacco via transformation of protoplasts with Ti-plasmid DNA: cotransfer of T-DNA and non T-DNA sequences. Plant Mol. Biol. 5:223–234.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Mariotti, D., Davey, M.R., Draper, J., Freeman, J.P., and Cocking, E.C. 1984. Crown gall tumorigenesis in the forage legume Medicago saliva L. Plant Cell Physiol. 25:473–482.

    CAS  Google Scholar 

  34. Herrera-Estrella, L., DeBlock, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M., and Schell, J. 1983. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 2:987–995.

    Article  CAS  Google Scholar 

  35. Bevan, M.W., Flavell, R.B., and Chilton, M.-D. 1983. A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187.

    Article  CAS  Google Scholar 

  36. Shillito, R.D., Saul, M.W., Paszkowski, J., Muëller, M., and Potrykus, I. 1985. High efficiency direct gene transfer to plants. Bio/Technology 3:1099–1103.

    Google Scholar 

  37. Deshayes, A., Herrera-Estrella, L., and Caboche, M. 1985. Liposome-mediated transformation of tobacco mesophyll protopolasts by an Escherichia coli plasmid. EMBO J. 4:2731–2737.

    Article  CAS  Google Scholar 

  38. Orr, W., Singh, J., and Brown, D.C.W. 1985. Induction of freezing tolerance in alfalfa cell suspension cultures. Plant Cell Rep. 4:15–18.

    Article  CAS  Google Scholar 

  39. Atanassov, A., and Brown, D.C.W. 1984. Plant regeneration from suspension culture and mesophyll protopolasts of Medicago sativa L. Plant Cell Tissue Organ Culture 3:149–162.

    Article  Google Scholar 

  40. Gamborg, O.L., Miller, R.A., and Ojima, K. 1968. Nutrient requirement of suspension culture of soybean root cells. Exp. Cell Res. 50:151–158.

    Article  CAS  Google Scholar 

  41. Currier, T.C., and Nester, E.W. 1976. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76:431–441.

    Article  CAS  Google Scholar 

  42. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning, Cold Spring Harbour Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  43. Graessmann, A., Graessmann, M., and Muëller, C. 1980. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 6:816–825.

    Article  Google Scholar 

  44. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Techn. 1:3–8.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biology, Carleton University, Ottawa, Ontario, K1S 5B6

    T. J. Reich & V. N. Iyer

  2. Genetic Engineering Section, Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K1A OC6, Canada

    B. L. Miki

Authors
  1. T. J. Reich
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  2. V. N. Iyer
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  3. B. L. Miki
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Reich, T., Iyer, V. & Miki, B. Efficient Transformation of Alfalfa Protoplasts by the Intranuclear Microinjection of Ti Plasmids. Nat Biotechnol 4, 1001–1004 (1986). https://doi.org/10.1038/nbt1186-1001

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  • DOI: https://doi.org/10.1038/nbt1186-1001

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