Theoretical and Applied Genetics

, Volume 82, Issue 2, pp 161–168

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

  • M. Battraw
  • T. C. Hall
Originals

DOI: 10.1007/BF00226207

Cite this article as:
Battraw, M. & Hall, T.C. Theoret. Appl. Genetics (1991) 82: 161. doi:10.1007/BF00226207

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-GlucuronidaseGene copy numberneo-mycin phosphotransferaseProtoplastsSorghum bicolor

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
  2. 2.Rhône-Poulenc AgrochimieLyonFrance