Efficient regeneration of protoplasts from cultivar Micro-Tom.

Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato () cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 10 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in species.