Factors affecting PEG-mediated stable transformation of maize protoplasts

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 11 ratio to 65% at a 1001 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.Abbreviations PEG polyethylene glycol - BMS Black Mexican Sweetcorn - MS salts of Murashige and Skoog (1962) culture medium - MaCa 0.2M mannitol, 80mM calcium chloride - MaMg 0.4M mannitol, 15mM magnesium chloride, 0.1% MES, pH 5.6 - CM conditioned medium - SE standard error of the mean