Nodal culture for efficient regeneration and CRISPR/Cas-based genome editing in recalcitrant horticultural crops

Abstract Nodal culture is a powerful plant tissue culture technique addressing critical challenges such as desiccation, microbial contamination, and the limited viability of explants, particularly in recalcitrant horticultural crops like Garcinia mangostana , Artocarpus heterophyllus , Cucumis melo , Citrus limon , Kinnow mandarin , and Coffea arabica . This method utilizes sterilized immature nodal explants, with regeneration induced through the precise application of growth regulators, primarily auxins and cytokinins, to media such as Driver-Kuniyuki (DKW), Woody Plant Media (WPM), and Murashige and Skoog (MS) under controlled conditions. These regulators significantly enhance both shoot and root regeneration, thus reducing the generation time for difficult-to-regenerate species. Reactive oxygen species (ROS) play a pivotal role in regulating cell division and hormone signaling during regeneration. Additionally, transcription factors such as wound-induced dedifferentiation 1 (WIND1), WUSCHEL (WUS), Enhancer of Shoot Regeneration 1 (ESR1), Cup-shaped Cotyledon 1 and 2 (CUC1, CUC2), and Lateral Organ Boundaries Domain 16 (LBD16) are integral to callus induction and organogenesis. Genetic variation observed in regenerated populations reflects the complexity of these regulatory networks and underscores the need for further investigation. Notably, nodal culture provides a promising alternative to conventional tissue culture methods, particularly in facilitating CRISPR/Cas9-mediated genetic modifications in recalcitrant crops. This technique enhances the efficient regeneration of transgenic horticultural crops, overcoming significant barriers to transformation. Future research should focus on refining nodal culture protocols across a broader spectrum of horticultural species, improving gene editing efficiency, and integrating this approach with advanced breeding technologies for targeted trait development and sustainable crop improvement.