Picrorhiza (Picrorhiza kurrooa) is an endangered medicinal plant with well-known hepatoprotective activity attributed to monoterpenoid picrosides. The present article details on regulatory genes of terpenoid metabolism, 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) and 1-deoxy-D-xylulose-5-phosphate synthase (pkdxs) from picrorhiza. Since no molecular information was available, these genes were cloned to full-length by degenerate primers and rapid amplification of cDNA ends, followed by cloning of the upstream sequences that showed the presence of core sequences for light and temperature responsiveness. Electrophoretic mobility shift assay confirmed binding of protein to these motifs. Expression of pkhmgr and pkdxs was up-regulated at 15 degrees C as compared to at 25 degrees C as well as under light as compared to dark conditions. Picrosides content exhibited the trend similar to gene expression. To rule out the possible limitation of carbon pool under dark condition, plantlets of picrorhiza were raised in vitro in Murashige and Skoog medium supplemented with 3% sucrose. Results showed similar up-regulation of both the genes and the higher picrosides content in in vitro raised plantlets in the presence of light. Data suggested the important roles played by light and temperature in regulating pkhmgr and pkdxs, and the picrosides level in picrorhiza.
The present study describes the effects of season on initial bud sprouting, direct shoot regeneration from the base of the sprouted bud, and cormlet production from multiple shoots, and provides growth evaluation of in vitro produced cormlets under greenhouse conditions. Initial sprouting of buds from corm segments was previously described in medium supplemented with 2,4‐dichlorophenoxyacetic acid (2,4‐D, 9.05 μM) and 6‐benzylaminopurine (BAP, 26.64 μM). Maximum bud sprouting (90%) was observed during November and December. Direct multiple shoot primordia were initiated from the base of these sprouted buds on BAP (26.64 μM). Multiplication of shoots was achieved in BAP (26.64 μM) and α‐naphthalene acetic acid (1.0 and 5.0 μM). Growth retardants (chlorocholine chloride and paclobutrazol) were used for cormlet production from multiple shoots, and paclobutrazol (1.7 μM) evinced maximum cormlet production (86.07%). Growth of these in vitro‐produced cormlets was evaluated under greenhouse conditions, and 91.66% sprouting was observed. An increase in cormlet weight (66.88%) was also observed under in vivo conditions.
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