Establishment of a seedling-based regeneration system for Taiwan oil millet (Eccoilopus formosanus) to support crop domestication and C4 biology

Taiwan oil millet (TOM; Eccoilopus formosanus) is a semi-domesticated orphan crop with potential as a resilient crop species and a model for C4 biology. However, the lack of an efficient tissue culture system has limited its use in genetic and functional studies. In this study, we developed a regeneration system for TOM using seedlings as the starting material for callus induction. Based on insights from previously reported sorghum tissue culture systems, we optimized seed sterilization, callus induction, and regeneration conditions, and further improved regeneration efficiency by incorporating plant growth regulators (PGRs). Efficient callus induction was achieved within four weeks, and the reduced calli maintained sustained proliferative capacity through repeated subculturing. The optimized protocol achieved a regeneration efficiency of 97.9%, with complete plant development from callus induction to mature, soil-acclimatized plants within 12–14 weeks. The use of seedlings provided a simple, accessible and scalable alternative to immature embryos, allowing year-round experimentation without strict developmental-stage constraints. In addition, TOM calli displayed stable regenerative capacity over extended culture periods. We established a robust and reproducible regeneration system from seedlings for TOM. This protocol provides a foundation for the future development of transformation and genome editing system in TOM, supporting its potential use in functional genomic studies and C4 biology. An efficient reproducible tissue culture system was established for Taiwan oil millet with the potential as a technique for crop domestication and C4 biology.