Theoretical and Applied Genetics

, Volume 82, Issue 2, pp 161–168 | Cite as

Stable transformation of Sorghum bicolor protoplasts with chimeric neomycin phosphotransferase II and β-glucuronidase genes

  • M. Battraw
  • T. C. Hall
Originals

Summary

Parameters influencing the stable transformation of Sorghum bicolor protoplasts with a chimeric neomycin phosphotransferase II (NPT II) gene by electroporation were investigated. The mean number of kanamycin-resistant calli produced increased in direct proportion to the concentration of DNA used for transformation. Linearization of the plasmid doubled the mean number of kanamycin-resistant calli produced, while the addition of carrier DNA had no effect. The copy number (1–4) of integrated genes was low compared with that frequently reported for PEG-mediated transformation. Two strategies for transforming protoplasts with a nonselectable, β-glucuronidase (GUS) gene were compared. One utilized a plasmid containing a CaMV 35S-NPT II gene covalently linked to a CaMV 35S-GUS gene, and the other strategy utilized the two genes on separate plasmids. DNA from all 77 kanamycin-resistant calli analyzed contained restriction fragments hybridizing to the NPT II probe; approximately 70% of the clones from all transformation treatments contained a 1.7-kb EcoRI/HindIII restriction fragment corresponding to the full-length gene. Of the kanamycin-resistant calli, 38–63% (depending on the transformation treatment) contained GUS-hybridizing fragments, and 8–19% contained the full-length gene. The addition of NPT II and GUS genes on a single plasmid or on separate plasmids did not appear to lead to an appreciable difference in the frequency of cointegration of these genes, although an increased proportion of the plasmid bearing the nonselectable (GUS) gene appeared to favor its cointegration.

Key words

β-Glucuronidase Gene copy number neo-mycin phosphotransferase Protoplasts Sorghum bicolor 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Battraw MJ, Hall TC (1990) Histochemical analysis of CaMV 35S promoter-β-glucuronidase gene expression in transgenic rice plants. Plant Mol Biol 15:527–538Google Scholar
  2. Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Gene Dev 1:1183–1200Google Scholar
  3. Chourey PS, Sharpe DZ (1985) Callus formation from protoplasts of Sorghum cell suspension cultures. Plant Sci 39:171–175Google Scholar
  4. Chourey PS, Zurawski DB (1981) Callus formation from protoplasts of a maize cell culture. Theor Appl Genet 59:341–344Google Scholar
  5. Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82:5824–5828Google Scholar
  6. Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793Google Scholar
  7. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant cell 2:603–618Google Scholar
  8. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  9. Jefferson RA, Kavanagh TA, Beven MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  10. Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74Google Scholar
  11. Labarca C, Paigen K (1980) A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352PubMedGoogle Scholar
  12. Laurie DA, Bennett MD (1985) Nuclear DNA content in the genera Zea and Sorghum. Intergeneric, interspecific, and intraspecific variation. Heredity 55:307–313Google Scholar
  13. Lyznik LA, Ryan RD, Ritchie SW, Hodges TK (1989) Stable cotransformation of maize protoplasts with gusA and neo genes. Plant Mol Biol 13:151–161Google Scholar
  14. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NYGoogle Scholar
  15. Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3:2717–2722Google Scholar
  16. Potrykus I (1990) Gene transfer to cereals: an assessment. Bio/Technol 8:535–542Google Scholar
  17. Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer to cells of a graminaceous monocot. Mol Gen Genet 199:183–188Google Scholar
  18. Potter H, Weir L, Leder P (1984) Enhancer-dependent expression of human χ immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 81:7161–7165Google Scholar
  19. Reed KC (1988) Evaluation of alkaline Southern transfers. Molecular Biology Rep 4:3–4Google Scholar
  20. Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221Google Scholar
  21. Reiss B, Sprengel R, Will H, Schaller H (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30:211–218Google Scholar
  22. Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988) Genetically transformed maize plants from protoplasts. Science 240:204–207Google Scholar
  23. Schocher RJ, Shillito RD, Saul MW, Paszkowski J, Potrykus I (1986) Cotransformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technol 4:1093–1096Google Scholar
  24. Schreier PH, Seftor EA, Schell J, Bohnert HJ (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J 4:25–32Google Scholar
  25. Shillito RD, Saul MW, Paszkowski J, Müller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Bio/Technol 3:1099–1103Google Scholar
  26. Shimamoto K, Terada RT, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274–276Google Scholar
  27. Taylor B, Powell A (1982) Isolation of plant DNA and RNA. Focus 4:4–6Google Scholar
  28. Uchimiya H, Fushimi T, Hashimoto H, Harada H, Syono K, Sugawara Y (1986 a) Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.). Mol Gen Genet 204:204–207Google Scholar
  29. Uchimiya H, Hirochika H, Hashimoto H, Hara A, Masuda T, Kasumimoto T, Harada H, Ikeda J-E, Yoshioka M (1986b) Coexpression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts. Mol Gen Genet 205:1–8Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • M. Battraw
    • 1
  • T. C. Hall
    • 1
  1. 1.Department of BiologyTexas A & M UniversityCollege Station, TXUSA

Personalised recommendations