Two Optimized Methods for Efficient, Stable and Transient Transformation of Broccoli (Brassica oleracea Var. Italica)

Broccoli (<i>Brassica oleracea</i> var. italica) is an important crop valued for its nutritional and health-promoting properties, yet its biotechnological improvement is limited by low effectivity and genotype-dependent transformation protocols. The absence of reliable transient expression systems further constrains functional genomics and genome-editing applications. Here, we optimized regeneration and transformation protocols for different broccoli genotypes. Endoreduplication patterns in young tissues were analyzed by flow cytometry to identify suitable explants, and combinations of plant growth regulators were tested to develop an efficient organogenic medium. Stable transformation was achieved via <i>Agrobacterium tumefaciens</i> using nptII and eGFP markers. Cotyledons and hypocotyls up to day 7 showed similar endoreduplication patterns, with abundant 2n cells, but hypocotyls exhibited higher regeneration capacity. The optimized medium supported efficient organogenesis while maintaining diploidy. Transformation efficiency reached 10.4% in 'S1' and 2.8% in 'Naxos', highlighting genotype dependence. In parallel, a transient expression system was established using cotyledon-derived protoplasts and electroporation-mediated DNA delivery. GFP expression was confirmed through fluorescence microscopy, confocal imaging, and Western blotting. These protocols provide a robust toolkit for broccoli genetic manipulation, facilitating molecular biology studies in the native plant, functional genomics and genome-editing strategies, including CRISPR-based approaches.