An efficient mesophyll protoplast isolation, purification and PEG-mediated transient gene expression for subcellular localization in Chinese kale

Abstract

Abstract Transient gene expression serves as a valuable tool for gene functional study in plants. Here we reported protoplast isolation and purification from the plantlet young leaves of Chinese kale (Brassica oleracae var. alboglabra Bailey), and their transient gene expression with polyethylene glycol (PEG)-mediated transformation and subcellular localization of phytoene desaturase (BaPDS1). The procedures of isolation andtransformation ofmesophyll protoplasts derived from Chinese kale were optimized, and the influencing factors were analyzed. The results showed that the optimal protocol of protoplast isolation and purification was initialized by digestion in enzyme solution (2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol) for 9 h. After filtered through 400 mesh and centrifuged at 179 ×g for purification, the total yield of protoplast reached as high as 6.04 × 107 protoplasts g−1 fresh weight (FW) and the viability of the protoplasts was up to 95%. A maximum transformation efficiency of approximately 30% measured by using green fluorescent protein (GFP) as a detecting gene was obtained when PEG4000 was at a final concentration of 40% and transformation time was set to 15 min. In addition, the subcellular localization of BaPDS1 in Chinese kale was targeted to the chloroplast, confirming the efficiency and reliability of this transient transformation system. Taken together, an efficient protoplast isolation, purification and transformation system in Chinese kale was established in this study, laying a foundation for future research in molecular biology and gene function in Chinese kale and other Brassica vegetables.