ABSTRACT
The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection and DNA microinjection, highlighting also the critical steps associated with the method. Briefly, we will describe the efficient isolation of protoplasts from 3-month-old suspension calli collected at 14 days after cultured. Digestion of the calli with an optimal composition of enzyme solution yielded over 2 × 106 protoplasts/mL with the viability of more than 80%. The concentrations of DNA, PEG, and magnesium chloride and application of heat shock treatment are the crucial determinants for efficient PEG-mediated transfection. Using the optimal PEG transfection conditions, a transfection efficiency of more than 20% could be obtained. At the same time, protoplasts embedded in alginate layer cultured for 3 days and injected with 100 ng/µL of total DNA solution are the optimal factors for microinjection. We successfully regenerated the injected protoplasts to calli expressing green fluorescent protein (GFP) signals when cultured in optimal medium and cultivation procedures.
