Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

M. R. Abdollahi, H. Rajabi Memari, Andre J van Wijnen

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1 Scopus citations

Abstract

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24. °C, 38. °C and 55. °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38. °C and 55. °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55. °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2. μM gibberellic acid.

Original languageEnglish (US)
Pages (from-to)96-102
Number of pages7
JournalGene
Volume487
Issue number1
DOIs
StatePublished - Nov 1 2011
Externally publishedYes

Keywords

  • β-Glucuronidase
  • Brassica napus
  • Endogenous GUS activity
  • Microspore-derived embryo
  • Rapeseed

ASJC Scopus subject areas

  • Genetics

Cite this

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells : How to avoid interference with the Gus transgene in transformation studies. / Abdollahi, M. R.; Memari, H. Rajabi; van Wijnen, Andre J.

In: Gene, Vol. 487, No. 1, 01.11.2011, p. 96-102.

Research output: Contribution to journalArticle