Optimization of induction and hairy root culture establishment of Teucrium chamaedrys L

This research was aimed to investigate the effect of two infection methods (immersion and spray) and two Agrobacterium rhizogenes strains (strain A13 and GMI9534) to induce hairy roots on different explants (hypocotyl, cotyledon, leaf, and stem node) of Teucrium chamaedrys. Strain GMI9534 could not induce hairy roots in any of the explants, whereas strain A13 was only able to induce hairy roots in leaf and stem node explants. Infection by immersion method was more successful, with higher root induction efficiency (more than 73.3%), and leaf explants showed the highest induction frequency (83.3%). The transformed hairy root lines were confirmed by PCR using rolA and rolB gene-specific primers. Significant differences were shown among the 20 independent hairy root lines cultured on growth regulator-free MS solid medium for total root length and for the root branching. These variables were stable across subcultures and hence seven independent hairy root lines were selected based on these growth properties. Subsequently, the cultures for these hairy root lines were established in half-strength MS liquid medium to monitor their biomass production during a 90-day culture period. Considerable variations were observed in growth capacity among the lines. Line TC-HR-16 produced the highest fresh biomass (9100 mg/30 ml culture medium), a 455-fold increase over initial inoculum, during the 10-weeks culture period. The best-characterized hairy root lines, resulted in this study, can be used to improve the production of secondary metabolites of pharmaceutical values of T. chamaedrys.