Cross-protection and selectable marker genes in plant transformation

  • Sung H. Park
  • Susan C. Rose
  • Cecilia Zapata
  • Metinee Srivatanakul
  • Roberta H. Smith
Genetic Transformation/Somatic Cell Genetics

Summary

Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.

Key words

tobacco Agrobacterium tumefaciens phosphinothricin hygromycin selection pressure cross-protection 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Cao, J.; Duan, X.; McElroy, D., et al. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep. 11:586–591; 1992.CrossRefGoogle Scholar
  2. Christou, P.; Ford, T. L.; Kofron, M. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica, and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9:957–962; 1991.CrossRefGoogle Scholar
  3. Deblaere, R.; Reynaerts, A.; Hofte, H., et al. Vectors for cloning in plant cells. Methods Enzymol. 153:277–292; 1987.CrossRefGoogle Scholar
  4. DeBlock, M.; Botterman, J.; Vandewiele, M., et al. Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO 6:2513–2518; 1987.Google Scholar
  5. Dekeyser, R.; Claes, B.; Marichal, M., et al. Evaluation of selectable markers for rice transformation. Plant Physiol. 90:217–223; 1989.PubMedGoogle Scholar
  6. Dellaporta, S. L.; Wood, J.; Hicks, J. B. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1:19–21; 1983.Google Scholar
  7. Hiei, Y.; Ohta, S.; Komari, T., et al. Efficient transformation of rice (Oryza sativa L.) mediated byAgrobacterium and sequence analysis of boundaries of the T-DNA. Plant J. 6:271–282; 1984.CrossRefGoogle Scholar
  8. Horsch, R. B.; Fry, J.; Hoffmann, N. L., et al. A simple and general method for transferring genes into plants. Science 227:1229–1231; 1985.CrossRefGoogle Scholar
  9. Ingelbrecht, I.; VanHoudt, H.; Van Montagu, M., et al. Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc. Natl. Acad. Sci. USA 91:10502–10506; 1994.PubMedCrossRefGoogle Scholar
  10. Klee, H.; Horsch, R. B.; Rogers, S. G.Agrobacterium-mediated plant transformation and its further applications to plant biology. Annu. Rev. Plant Physiol. 38:467–486; 1987.CrossRefGoogle Scholar
  11. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  12. Ortiz, J. P. A.; Reggiardo, M. I.; Ravizzini, R. A., et al. Hygromycin resistance as an efficient selectable marker for wheat stable transformation. Plant Cell Rep. 15:877–881; 1996.CrossRefGoogle Scholar
  13. Park, S. H.; Pinson, S. R. M.; Smith, R. H. T-DNA integration into genomic DNA of rice followingAgrobacterium inoculation of isolated shoot apices. Plant Mol. Biol. 32:1135–1148; 1996.PubMedCrossRefGoogle Scholar
  14. Wan, Y.; Lemaux, P. G. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104:37–48; 1994.PubMedGoogle Scholar

Copyright information

© Society for In Vitro Biology 1998

Authors and Affiliations

  • Sung H. Park
    • 1
  • Susan C. Rose
    • 1
  • Cecilia Zapata
    • 1
  • Metinee Srivatanakul
    • 1
  • Roberta H. Smith
    • 1
  1. 1.Department of Soil and Crop SciencesTexas A&M UniversityCollege Station

Personalised recommendations