FACTORS AFFECTING TRANSIENT EXPRESSION OF VECTOR CONSTRUCTS IN WHEAT PROTOPLASTS

Direct uptake of reporter gene constructs with the bacterial beta-gluc oronidase (GUS) gene fused to various promoters was achieved to enbryo genic cell suspension culture-derived protoplasts of GK Sagvari winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment . Based on GUS specific activity values, it was found that Mg2+ with P EG 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 degrees C. Transient GUS expression as detected by spectrof luorimetry was positively correlated with the time elapsed after DNA u ptake (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 prot oplasts 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 pro moter plus two enhancer elements than with the other constructs tested .