Morphological and molecular variation in Ri-transformed root lines are stable in long term cultures of Tylophora indica

Abstract

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 Ritransformed root lines could be divided into two groupsGroup 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 A 4 20 and A 4 26 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.

The Fate of Integrated Ri T-DNA rol Genes during Regeneration via Somatic Embryogenesis in Tylophora indica

“…Thus, aux genes were neither essential for hairy root syndrome nor for spontaneous induction of embryogenic calli in T. indica. We have reported detailed study with 15 of these root lines, performed for over 4 years showing that the morphological, biochemical, and molecular variation in the Ri-transformed root lines were stable in long-term cultures in T. indica [29].…”

“…Fifty fast-growing Ri-transformed roots lines showing morphological characteristics typical of Ritransformed roots, such as high branching and rapid and plagiotropic growth on hormone-free media, were established and maintained as reported earlier [29]. However, the root lines showed low regeneration frequency (8/50 root lines).…”

“…Hairy root cultures of Tylophora indica were established with Agrobacterium rhizogenes strain A4 and maintained as reported earlier [29]. Fifty Ri-transformed root lines were cultured on MS medium (Murashige and Skoog's [30] salts supplemented with 3% (w/v) sucrose and 0.75% (w/v) agar (Sigma, India)) in dark and under photoperiod of 16/8 hours to study regeneration of plants from the hairy root cultures.…”

“…The RNA was quantified spectrophotometrically (BioSpectrometer, Eppendorf, Germany) and the same reaction procedure was used for the RT-PCR of all of the genes tested (the rolA, rolB, rolC, and rolD genes of TL-DNA and TR-DNA). The RT-PCR reactions were performed as described earlier [29]. Finally, PCR analysis was done with the prepared cDNA samples (2 L) following earlier described method [27,28,36].…”

“…Recently we have reported the stability of morphological and molecular variation of Ri-transformed root lines in long-term cultures of T. indica [29]. In order to investigate the fate of integrated Ri T-DNA rol genes during regeneration via indirect somatic embryogenesis and stability of its effect on Ri-transformed plants, we have characterised Ri-transformed root cultures, embryogenic calli, somatic embryos, and somatic embryo derived plants of T. indica.…”