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Many uses of protoplasts, plant cells with the cell wall removed, have been explored. Many advantages of the system have been realized and proven in recent years in various physiological, biochemical, genetic, and molecular biological studies. Reliable methods to isolate viable protoplasts from a broad variety of plant species have been established. Regeneration of plants from protoplasts has become one of the options involved in crop gene manipulation and crop improvement. Here, we present how protoplast system may help crop gene editing and novel trait development, and discuss the potentials and challenges of this approach.