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• Cotyledonary leaves, shoot tips, hypocotyls and nodes were used to accomplish regeneration methods in Spermacoce hispida. • High frequency plant regeneration through organogenesis and somatic embryogenesis was achieved from nodal segments. • Hypocotyl-derived calli were induced shoot regeneration on MS medium containing kinetin. • Survival rate of successfully accomplished plantlets was evaluated under greenhouse and at filed conditions. • Histological evidences confirmed the regeneration mode through organogenesis and somatic embryogenesis. Shaggy button weed (Spermacoce hispida L.) is a therapeutically important herb widely used in Indian System of medicine particularly Ayurvedic, Siddha, and Unani. S. hispida is recalcitrant during germination in natural habitat. Plant tissue culture is an alternate approach for mass multiplication of such vulnerable plants. Plant regeneration by organogenesis and somatic embryogenesis in S. hispida was attempted using cotyledonary leaf, shoot tip, hypocotyl, and nodal explants. Maximum shoot regeneration frequency (72%) and number of shoots (10.5 ± 0.3) through direct organogenesis was achieved from nodal explants cultured on Murashige and Skoog (MS) medium amended with 2.0 mg/dm3 of 6-benzylaminopurine (BAP). Further, MS medium containing 0.5 mg/dm3 2, 4-dichlorophenoxyacetic acid (2, 4-D) produced higher frequency of callus induction (86.25%) from hypocotyl segments. Optimum shoot regeneration response (69.5%) through indirect organogenesis was observed from hypocotyl-derived callus on MS medium fortified with 1.0 mg/dm3 kinetin with 6.1 shoots per callus. The highest regeneration frequency (68%) through somatic embryogenesis was recorded from nodal explants on MS medium supplemented with 2.0 mg/dm3 BAP and 0.8 mg/dm3 kinetin. The regenerated plantlets were acclimatized successfully under ex vitro conditions with normal phenotypic appearance. Shoot tips and nodes did not induce organogenesis (indirect), whereas nodal segments showed better regeneration via organogenesis (direct) and somatic embryogenesis in MS medium with either BAP or BAP with kinetin. Histological examinations confirmed the distinctive developmental stages and bipolar structure of somatic embryos. The described regeneration protocols can be effectively utilized for the large-scale multiplication, germplasm conservation, pharmacological and genetic manipulation studies. [ABSTRACT FROM AUTHOR]