Red and Far-Red LED Lighting Enhances Protoplast-to-Plant Regeneration in Broccoli (Brassica oleracea var. italica)

Plants have a remarkable ability to regenerate tissues and organs from single cells, a property that underpins in vitro protoplast regeneration. Efficient protoplast-to-plant regeneration remains a major bottleneck for genome engineering in many crop species, including broccoli (<i>Brassica oleracea</i> var. <i>italica</i>). In this study, we established and optimized a regeneration system for broccoli cv. Claremont by evaluating enzyme composition, light quality, and culture media at successive stages of development. Among the tested enzyme mixtures, 1.5% Cellulase R-10 combined with 0.4% Macerozyme R-10 yielded the highest protoplast viability and recovery. Alginate-embedded protoplasts were cultured under control (dark), blue, and red + far-red LED illumination. Red + far-red treatment significantly enhanced microcolony formation, plating efficiency, and shoot regeneration compared with blue light, whereas blue illumination consistently reduced regenerative performance. The inclusion of activated charcoal in the regeneration medium further increased shoot production. The generalized linear model analyses identified light quality as a significant predictor of both shoot number and regeneration. To our knowledge, this study provides one of the first demonstrations of LED-assisted enhancement of protoplast regeneration in broccoli. The optimized protocol enables whole-plant recovery within approximately 5 months and offers a practical platform for CRISPR-based genome editing and advanced breeding applications in <i>B. oleracea</i>.