Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

Abstract

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.

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

“…5 The hairy root cultures were maintained in 50 ml modified B5 liquid medium (3% sucrose) pH 5.8 in 250 ml Erlenmeyer flasks. The cultures were incubated on a shaker (90 rpm) under standard cool white fluorescent tubes with a flux rate of 35 mmols 16-h photoperiod at 25 § 2 0 C. The roots were regularly sub-cultured after a period of 4 weeks.…”

“…3 The medicinally important components of P. kurroa are picroside I, picroside II, picroside III, picroside IV, apocynin, androsin, catechol, and kutkoside. 3,5 Agrobacterium rhizogenes induced hairy root cultures have attracted much attention in recent years as advantageous resource of useful plant based compounds. 6 Hairy root culture has revolutionized the role of in vitro plant organ culture for the secondary metabolite production.…”

“…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.…”

“…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. We investigated the effect of different media composition at full and half strengths of salts with or without supplementation of the additional vitamin source with different concentrations and different carbon sources for the production enhanced potential both in terms of biomass and secondary metabolites during different growth phases of selected hairy root line (14-P) of P. kurroa.…”