Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes

Efficient regeneration protocols are essential for large-scale propagation and genetic manipulation of recalcitrant medicinal species such as Cannabis sativa. Existing direct and indirect regeneration methods are highly genotype and explant-dependent, limiting broader applicability. Here, we report a five-stage (S₀-S₄) optimised protocol that is reproducible and achieves high-efficiency direct de novo regeneration using cotyledonary node explants from both hemp and medicinal cannabis genotypes. A 1% (v/v) H₂O₂-based sterilisation method significantly improved seed germination and reduced endophyte contamination. Among embryo-derived explants, the cotyledonary node attached to the cotyledon showed superior regeneration efficiency through two distinct pathways: axillary shoot initiation and de novo regeneration, the latter achieving ~ 70-90% efficiency in six hemp cultivars and three medicinal cannabis lines on TDZ and NAA containing shoot regeneration medium. Histological analysis confirmed true de novo shoot formation from peripheral cortical cells, independent of pre-existing meristems or callus. De novo shoots were initiated within 2 d of shoot regeneration medium treatment, indicating rapid cellular commitment to organogenesis, with optimal regeneration between 7 and 14 d. Prolonged exposure proved detrimental, causing excessive callusing and vitrification. Repeated subculturing during proliferation stage enabled scalable shoot multiplication, yielding an average of 7 shoots per responding explant (~ 11.4 shoots per seed), outperforming previously published cotyledon-based (~ 2-fold) and hypocotyl-based (~ 5-fold) methods under comparable conditions. Regenerated plantlets developed healthy roots (with IAA or IBA) and acclimatised readily, exhibiting normal vegetative and reproductive growth. The protocol's reproducibility across diverse cannabis genotypes and its applicability to other medicinal angiosperm species in this study highlights its value for both research and commercial applications.