ENHANCED GUS GENE-EXPRESSION IN CEREAL GRASS CELL-SUSPENSIONS AND IMMATURE EMBRYOS USING THE MAIZE UBIQUITIN-BASED PLASMID PAHC25

Transient GUS (beta-glucuronidase) expression was visualized in cell s uspensions of Triticum aestivum, Zea mays, Pennisetum glaucum, Sacchar um officinarum, Pennisetum purpureum and Panicum maximum after micropr ojectile bombardment with pBARGUS and pAHC25 plasmid DNAs. pBARGUS con tains the GUS (UidA) gene coding region driven by the Adh1 promoter an d the Adh1 intron 1, as well as the BAR gene coding region driven by t he CaMV 35S promoter and the Adh1 intron 1. pAHC25 contains the GUS an d BAR gene coding regions driven by the maize ubiquitin promoter, firs t exon and first intron (Ubi1). The effectiveness of the constructs wa s first compared in cell suspension cultures by counting blue expressi on units (b.e.u.). The expression of construct pAHC25 ranged from 3 to 50 fold greater than pBARGUS in different species. In addition, the t wo plasmids were quantitatively compared in Triticum aestivum and Zea mays by using the more sensitive GUS fluorometric assay to determine t he amount of methylumbellyferride (MU) produced. There was more than a 30 fold increase in MU production with pAHC25 than with pBARGUS in th e 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 bombard ment with pBARGUS and pAHC25 DNA. Expression of plasmid pAHC25 was twi ce as high as pBARGUS. A comparison of two DNA/gold preparation method s, as well as repeated sonications of the DNA/gold mixture, had no eff ect on the number of b.e.u.