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. 1988 Feb;86(2):602–606. doi: 10.1104/pp.86.2.602

Evaluation of Selectable Markers for Obtaining Stable Transformants in the Gramineae 1

R M Hauptmann 1,2, V Vasil 1,2, P Ozias-Akins 1,2, Z Tabaeizadeh 1,2, S G Rogers 1,2, R T Fraley 1,2, Robert B Horsch 1,2, Indra K Vasil 1,2,2
PMCID: PMC1054530  PMID: 16665953

Abstract

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10−5 to 2 × 10−6) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Eichholtz D. A., Rogers S. G., Horsch R. B., Klee H. J., Hayford M., Hoffmann N. L., Braford S. B., Fink C., Flick J., O'Connell K. M. Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants. Somat Cell Mol Genet. 1987 Jan;13(1):67–76. doi: 10.1007/BF02422300. [DOI] [PubMed] [Google Scholar]
  3. Fromm M. E., Taylor L. P., Walbot V. Stable transformation of maize after gene transfer by electroporation. 1986 Feb 27-Mar 5Nature. 319(6056):791–793. doi: 10.1038/319791a0. [DOI] [PubMed] [Google Scholar]
  4. Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5824–5828. doi: 10.1073/pnas.82.17.5824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lloyd A. M., Barnason A. R., Rogers S. G., Byrne M. C., Fraley R. T., Horsch R. B. Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science. 1986 Oct 24;234(4775):464–466. doi: 10.1126/science.234.4775.464. [DOI] [PubMed] [Google Scholar]
  6. Ou-Lee T. M., Turgeon R., Wu R. Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6815–6819. doi: 10.1073/pnas.83.18.6815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  8. Shure M., Wessler S., Fedoroff N. Molecular identification and isolation of the Waxy locus in maize. Cell. 1983 Nov;35(1):225–233. doi: 10.1016/0092-8674(83)90225-8. [DOI] [PubMed] [Google Scholar]

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