TRANSIENT GENE-EXPRESSION SYSTEM USING PROTOPLASTS FROM DEVELOPING SOYBEAN COTYLEDONS

A procedure for isolating protoplasts from developing soybean cotyledons was developed. Electroporation conditions for transient expression of the beta-glucuronidase (GUS) gene introduced into protoplasts of soybean cotyledons from Glycine max cv. Raiden were determined. The factors studied included electric field strength (EFS), pulse length and protoplast concentration, carrier DNA and plasmid DNA concentrations, divalent cations and incubation time. The optimum conditions were found to be as follows: one or two pulses each 6.6 ms in length at 800 V/cm; a protoplast concentration of 3.6 x 10(5) viable cells/ml; a carrier DNA concentration of 20 approximately 40 mug/ml; a plasmid DNA concentration of 10 approximately 20 mug/ml; an electroporation buffer containing 0.1 mM CaCl2; and an incubation time of 48 approximately 96h. This system would be available for the construction and introduction of chimeric genes, and assaying their expression for the improvement of soybean seed quality.