Agrobacterium Rhizogenes-mediated Transformation of Peganum multisectum (Maxim) Bobrov and Harmine Production in Hairy Roots

Abstract

Using Agrobacterium rhizogenes due to create hairy roots is a useful method to product secondary metabolites in many medicinal plants. The transgenic hairy roots were induced from Peganum multisectum (Maxim) Bobrov a medicinally important species, by infecting leaf and stem explant with wild type Agrobacterium rhizogenes strain ATTCC 15834, which led to the induction of hairy roots from 19% of the explants. Polymerase chain reaction with primers for rol A confirmed the integration of T-DNA fragment of Ri plasmid of A. rhizogenesis into the genome of hairy roots obtained after transformation. Four transformed lines of hairy roots were established. Doubling time of the faster growing hairy root lines was about 11 days and these cultures showed about 12-fold increase in biomass at the end of 6 weeks as compared to non-transformed roots. The hairy roots showed an ability to synthesize harmine, a β-carboline alkaloid of medicinal value. The effect of the initial sucrose and ammonium nitrate concentration in biomass and harmine production of the liquid MS cultural medium cultures was studied. The highest values for harmine accumulation and fresh weight were obtained between 30-45 g l-1 of sucrose. The results also showed that the addition of extra ammonium nitrate up to optimum level (2475 mg l-1), as a source of nitrogen was significantly effective than standard ammonium nitrate in MS basal medium for growth of hairy roots and harmine production. This is the first report on the induction of hairy roots in P. multisectum (Maxim) Bobrov.