Organogenesis, direct somatic embryogenesis, and shoot proliferation of Rheum spiciforme Royle: an endemic and vulnerable medicinal herb from Indian Trans Himalayas

Rheum spiciforme Royle is a high value medicinal herb restricted to NW Himalayas. The medicinal properties of Rheum include anti-oxidant, anti-microbial, antitumor, anti-inflammatory, anti-fungal, anti-atherosclerotic, anti-proliferative, hepatoprotective, and immuno-enhancing. The species is threatened and endemic which demands its conservation. In this context, we have developed a premiere efficient in vitro regeneration system for this herb. The seed germination displayed phenomenal increase when transferred from soil (13.6 +/- 3.1%) to half-strength Murashige and Skoog (MS) medium (92.6 +/- 1.3%) fortified with 0.005 mM gibberellic acid (GA(3)) and 1 mM potassium nitrate (KNO3) and calcium chloride (CaCl2) each with mean germination time (MGT) of 8.5 +/- 1.8 d. Among four types of explants used for callusing, leaf explants responded highest with 87.3 +/- 1.4% at 2 mu M 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) each. Nodal-segment-derived brown calluses exhibited significantly high regeneration (96.3 +/- 1.6 %) at 8.0 mu M BA and kinetin (KIN) each with 4.0 mu M GA(3). Leaf explants observed direct somatic embryogenesis which displayed maximum (91.0 +/- 3.4%) germination at 25 mu M BA, 1.0 mu M NAA (naphthaleneacetic acid), 2.0 mu M GA(3), 50.0 mu M glutamine (GTM), and adenine sulfate (ADS) each. Multiple shoot induction with mean number of 10.1 +/- 2.6 shoots and elongation 4.2 +/- 0.4 cm was observed at 12.5 mu M BA and 0.5 mu M NAA along with 25 mu M GTM and ADS each. The rooted seedlings developed in half-strength liquid MS with 2.5 mu M NAA were hardened and subsequently transferred to the field. The developed protocol could be utilized for various attributes which include development of large-scale micropropagation system as a conservation measure, Agrobacterium-mediated genetic transformation studies, and industrial production of important bioactive chemical constituents.

Automated summary

A highly efficient in vitro regeneration system was developed for Rheum spiciforme Royle, resulting in significant increase in seed germination rates with the use of plant hormones in the medium. Different explants showed varying responses to regeneration conditions, leading to successful cultivation and transplantation.