Optimization of conditions for electroporation and transient expression of foreign genes in sugarcane protoplasts

Abstract

Electroporation conditions were established suitable for consistent high level transient expression of introduced reported genes in protoplasts of sugarcane (interspecific hybrids of Saccharum spp.) with minimal effect on protoplast survival. Factors investigated included the electrical conditions, buffer pH and conductivity, and membrane stabilizers. Level of expression was highly dependent on the electrical conditions, with electrical field strength being critical. Inclusion of 5 mM Ca 2+ in the electroporation buffer was essential for protoplast survival. Conditions optimized using one cultivar, Q63, were readily adapted to a second cultivar, Q96, indicating that the procedure can probably be extended rapidly to new sugarcane cultivars. Quality of protoplasts was important, affecting both the level of expression and optimum electrical conditions. The conditions determined in this study will be useful for work such as promoter testing requiring an effective transient expression system, and also provide an ideal starting point for establishing a procedure for stable transformation in sugarcane.