Optimization of polyethylene glycol mediated transient gene expression in pea protoplasts

We have investigated factors influencing polyethylene glycol mediated DNA uptake and ß-glucuronidase expression in pea (Pisum sativum L.) protoplasts. It was found that for optimal \-glucuronidase expression the molecular weight and concentration of polyethylene glycol should be 4000 and 20%, respectively. The amount of plasmid DNA should be 25 g per 5×10⁵ protoplasts in each treatment, and the concentration of Mg²⁺ in the transformation buffer should be 15 mM. The optimized protocol was applicable to all four pea cultivars tested.Abbreviations FDA fluorescein diacetate - GUS ß-glucuronidase - MU 4-methylumbelliferone - MUG 4-methyl umbelliferyl glucuronide - MW molecular weight - PEG polyethylene glycol - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide