Efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in greater yam (<i>Dioscorea alata</i>)

Abstract

Greater yam (Dioscorea alata) provides staple food for more than 100 million people in tropical and sub-tropical countries. Developing transient transformation platform is critical for gene function analysis, since huge amount of genomic and transcriptomic data for greater yam is recently available. In this study, we determined the best enzyme combination of 0.8% cellulase and 1% macerozyme and 6 hours of digestion to get greater yam protoplast output as 3.7 × 10⁷ protoplasts per gram of fresh leaf and 92% viable protoplasts. PEG-mediated transient transformation efficiency for greater yam was 66.2% with optimized PEG concentration (20%) for 20 min. Using this protocol, the subcellular location of a transcription factor in greater yam - DaERF2 was specifically in nuclei. This efficient protoplast isolation and transformation protocol provides feasible system for protein subcellular localization, and many other molecular assays in characterizing gene functions.