Enhanced GUS gene expression in cereal/grass cell suspensions and immature embryos using the maize uhiquitin-based plasmid pAHC25

Abstract

Transient GUS (β-glucuronidase) expression was visualized in cell suspensions of Triticum aestivum, Zea mays, Pennisetum glaucum, Saccharum officinarum, Pennisetum purpureum and Panicum maximum after microprojectile bombardment with pBARGUS and pAHC25 plasmid DNAs. pBARGUS contains the GUS (UidA) gene coding region driven by the Adh1 promoter and the Adh1 intron 1, as well as the BAR gene coding region driven by the CaMV 35S promoter and the Adh1 intron 1. pAHC25 contains the GUS and BAR gene coding regions driven by the maize ubiquitin promoter, first exon and first intron (Ubi1). The effectiveness of the constructs was first compared in cell suspension cultures by counting blue expression units (b.e.u.). The expression of construct pAHC25 ranged from 3 to 50 fold greater than pBARGUS in different species. In addition, the two plasmids were quantitatively compared in Triticum aestivum and Zea mays by using the more sensitive GUS fluorometric assay to determine the amount of methylumbellyferride (MU) produced. There was more than a 30 fold increase in MU production with pAHC25 than with pBARGUS in the wheat suspension, while the maize suspension showed only a 2.5 fold increase with the pAHC25 construct. Transient GUS expression was also visualized in immature embryos of Pennisetum glaucum following bombardment with pBARGUS and pAHC25 DNA. Expression of plasmid pAHC25 was twice as high as pBARGUS. A comparison of two DNA/gold preparation methods, as well as repeated sonications of the DNA/gold mixture, had no effect on the number of b.e.u.