Morphological and molecular variation in Ri-transformed root lines are stable in long term cultures of Tylophora indica
Agrobacterium rhizogenes (Ri) transformed root lines of Tylophora indica were established and characterized on the basis of morphology, insertion and expression of T-DNA genes, DNA profiling and tylophorine content in order to study the stability of Ri-transformed root lines in long term culture. Morphologically Ri-transformed root lines were of four phenotypes—moderately branched thin roots, moderately branched thick roots, highly branched thin roots and highly branched thick roots. On the basis of the presence and expression of different T-DNA genes, the Ri-transformed root lines could be divided into two groups—Group I (13 %): TL+/TR+ and Group II (87 %): TL+/TR−. The presence and expression of TR-DNA in addition to TL-DNA did not have any effect on the root morphology and tylophorine content. Tylophorine content varied from 1.01 ± 0.05 to 1.25 ± 0.02 mg gDW−1 in 15 Ri-transformed root lines studied. The transformed root lines stably retained their characteristic phenotype, growth rate, integration and expression of T-DNA genes and tylophorine accumulation potential in long term culture i.e., for 4 years. None of the root lines showed any difference in DNA fingerprinting profiles for each of the 11 OPA primers, showing genetic stability and clonal fidelity of the root lines and root clones in long term culture. Transformed root lines A420 and A426 showing high tylophorine content (1.24 ± 0.03 and 1.22 ± 0.05 mg gDW−1 respectively) even after 4 years of maintenance in vitro on phytohormone unsupplemented medium can be used for scale up studies. This work endorses the utility of T. indica transformed roots for the production of secondary metabolites in bioreactors.