Efficient plant regeneration and histological evaluations of regenerants through organogenesis and somatic embryogenesis in Spermacoce hispida L.-An underutilized medicinally important plant

摘要

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/dm(3) of 6-benzylaminopurine (BAP). Further, MS medium containing 0.5 mg/dm(3) 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/dm(3) 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/dm(3) BAP and 0.8 mg/dm(3) 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.