Factors affecting transient expression of vector constructs in wheat protoplasts

K. Z. Ahmed, S. Omirulleh, F. Sági, D. Dudits

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Direct uptake of reporter gene constructs with the bacterial β-glucoronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca2+-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25°C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

Original languageEnglish
Pages (from-to)209-220
Number of pages12
JournalActa biologica Hungarica
Volume48
Issue number2
Publication statusPublished - Dec 1 1997

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Keywords

  • Protoplast
  • Reporter gene
  • Triticum aestivum
  • Vector

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Environmental Science(all)
  • Neurology

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