In vitro regeneration of Aristolochia tagala and production of artificial seeds

Protocols for in vitro plant multiplication from somatic tissues and production of artificial seeds through encapsulation of nodes were developed for Aristolochia tagala Cham., a rare and valuable medicinal plant, as a measure of conservation and as a prerequisite for genetic transformation procedure. A maximum number of adventitious shoots were regenerated from leaf-derived callus on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 2 μM), α-naphthaleneacetic acid (NAA; 0.5 μM), and phloroglucinol (PG; 10μM). Nodes collected from in vitro established shoot cultures were encapsulated in 3 % (m/v) sodium alginate and 1 % (m/v) calcium chloride. Multiple shoots were successfully regenerated from the encapsulated nodes cultured on MS medium supplemented with 3 μM BAP and 0.5 μM kinetin (KIN). Regenerated shoots from callus and artificial seeds were successfully rooted and acclimated to greenhouse conditions. Since roots of A. tagala are primarily used in traditional medicine, a protocol for regenerating roots directly from the leaf derived callus was also developed. Maximum root length was obtained when the callus was cultured in MS medium supplemented with KIN (1 μM), indole acetic acid (IAA; 0.5 μM), NAA (0.1 μM), and PG (10 μM). Biochemical parameters were studied in calli grown with and without PG in the medium to establish a correlation between these parameters and shoot morphogenesis. An increment of antioxidant enzymes (peroxidase and catalase) and metabolites (sugars and proteins), and a decrease in the amount of polyphenol oxidase was observed in the calli which were grown in the presence of PG.