Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

Abstract

The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 × 106 protoplasts g−1 FW) and a survival rate of > 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min−1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for 25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane.