Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g-1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.

Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.