Transient expression of electroporated gene in leaf protoplasts of indica rice and influence of template topology and vector sequences

Abstract

Electroporation was used for gene delivery and evaluation of various parameters affecting transient expression of a gene for β-glucuronidase (gus) in leaf protoplasts of Oryza sativa var. Basmati-370. Transient expression was found to be dependent on voltage, capacitance, amount of plasmid and carrier DNA as well as period of culture. Maximum GUS activity was obtained when a 150 ms pulse at 300 V cm^-1 and 200 μF was applied to the protoplasts (l-2×10⁶ml^-1) in an electroporation buffer containing 20 μg of plasmid and 30 μg of calf thymus DNA, assayed 48 h after electroporation. DNA topology did not influence expression of the gene as both linear and supercoiled templates resulted in similar activities, but a 4-fold decrease in expression was observed if the gene was excised, reflecting the positive influence of vector sequences on gene expression.