Hairy root culture of Picrorhiza kurroa Royle ex Benth.: a promising approach for the production of picrotin and picrotoxinin

Abstract

Picrorhiza kurroa Royle ex Benth. is an endangered plant producing various compounds of medicinal importance. Hairy roots of P. kurroa were obtained following cocultivation of shoot tip explants with Agrobacterium rhizogenes strains A 4 and PAT 405. Bacterial strain A 4 appeared to be better than the strain PAT 405 in terms of both growth of respective hairy root cultures and secondary metabolite production. The optimal growth of both the hairy root cultures occurred on halfstrength semisolid medium with 3% sucrose. Picrotin and picrotoxinin from the roots of wild type field grown plants were compared with 8-week-old hairy root cultures induced by the A 4 and PAT 405 strains of A. rhizogenes. Picrotin and picrotoxinin content were evaluated in hairy root cultures as well as roots of field grown plant of P. kurroa. In terms of the production of picrotin and picrotoxinin, the A 4 induced hairy roots appeared to be a better performer than the PAT 405 induced hairy root cultures. The picrotin and picrotoxinin content was highest in 8-week-old A 4 induced hairy roots (8.8 lg/g DW and 47.1 lg/g DW, respectively). Rapid growth of the hairy roots of P. kurroa with in vitro secondary metabolite production potential may offer an attractive alternative to the exploitation of this endangered plant species.

Effect of elicitation on picrotin and picrotoxinin production from in vitro products of Picrorhiza kurrooa

“…The fresh weight and dry weight after lyophilization were determined determined. The powdered samples (0.5 g DW) were extracted with 100 ml of 70% ethanol in a soxhlet apparatus, and then dried crude extract was dissolved in 10 ml of water:methanol:isopropanol: acetonitrile (60:30:5:5) and quantification by HPLC was done using standards (Purohit et al, 2008;Mishra et al, 2011b). A standard curve was prepared by 10, 20, 30, 40 and 50 mg/L solutions of picrotin and picrotoxin standard (Sigma Chemical Company, St. Louis, USA).…”

“…Detection was done at 220 nm using UV detector L 7400. The calculations of picrotin and picrotoxinin were carried out on peak area basis using standard curves made with reference standard compound (Mishra et al 2011b). …”

“…Production of picrotin and picrotoxinin from plant tissue cultures is important because intact plants produce this compound only in small amounts. The reported yield of active ingredients (picrotin and picrotoxinin) from field-grown plant of P. kurrooa is 0.64 and 7.49 µg/g dry weight (DW) (Mishra et al, 2011b). Several papers have been published on micropropagation (Lal et al, 1988;Chandra et al, 2004;Chandra et al, 2006, Sood andChauhan, 2011), synthetic seed formation (Mishra et al, 2011a), hairy root culture and regeneration from hairy roots of P. kurrooa (Mishra et al, 2011b;Rawat et al 2013), however, assessment of secondary metabolites, that is, picrotin and picrotoxinin in tissue culture raised plants of P. kurrooa has not reported so far.…”

Yield enhancement strategies for the production of picroliv from hairy root culture ofPicrorhiza kurroaRoyle ex Benth.

“…8 The biosynthetic potential of the transformed roots is genetically controlled but it has frequently been observed that such transformed roots are sensitive to medium composition with respect to both biomass yield and secondary metabolite productivity. 5,9,10 Several groups have shown that besides selection of superior hairy root clones with higher than average productivity, media optimization could enhance the biomass as well as secondary metabolite yield. 5,10 We previously reported A. rhizogenes mediated transformation of this medicinally imperative plant species for the production of the desired phytochemicals (i.e., Picropside I and Kutkoside).…”

“…5,9,10 Several groups have shown that besides selection of superior hairy root clones with higher than average productivity, media optimization could enhance the biomass as well as secondary metabolite yield. 5,10 We previously reported A. rhizogenes mediated transformation of this medicinally imperative plant species for the production of the desired phytochemicals (i.e., Picropside I and Kutkoside). 5 In present study we optimized the different parameters to enhance productivity of picroliv from hairy roots of P. kurroa.…”