An efficient cucumber (Cucumis sativus L.) protoplast isolation and transient expression system

The transient gene expression system using plant protoplasts has become widely used for high-throughput analysis and functional characterization of genes. In this work we investigated protoplast isolation and green fluorescent protein (GFP) transient transfection and their main affecting factors, such as mannitol concentration in enzymolysis solution, enzymolysis time, and polyethylene glycol (PEG) concentration and transfection time, on 'xintaimici' cucumber. The results showed that when the enzyme solution had 1.5% cellulase R-10 (w/v), 0.4% macerozyme R-10 (w/v), 0.4M mannitol, 20 mM 2-morpholinoethanesulfonic acid, 10 mM CaCl2, 0.1% bovine serum albumin, and was at pH 5.8 with an enzymolysis time of 8 h, the protoplast yield was 6-7 x 10(6)/g fresh weight. Viability was about 90%. GFP was used as the reporter gene to measure protoplast transformation efficiency. When the concentration of PEG4000 was 20% and transfection time was 20 or 30 min, transformation efficiency was greater than 50% and the green fluorescent signal could be detected in the cytoplasm, chloroplasts, and plasma membrane. We show here an efficient PEG-mediated cucumber protoplast transient expression system using GFP reporter gene, laying a technical foundation for future research in cucumber molecular biology. (C) 2012 Elsevier B.V. All rights reserved.