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

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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 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.

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Authors and Affiliations

  1. Department of Botany, Centre of Advanced Study, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

    Dipasree Roychowdhury, Amrita Basu & Sumita Jha

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  1. Dipasree Roychowdhury
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  2. Amrita Basu
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  3. Sumita Jha
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Correspondence to Sumita Jha.

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10725_2014_5_MOESM1_ESM.tif

Fig. S1 Agarose [1.2 % (w/v)] gel electrophoresis of PCR products with rolA-, rolB-, rolC-, rolD- and TR-DNA specific primers. Lane 1: molecular markers (100 bp plus DNA ladder); Lane 2: positive control (pLJ1, containing TL-DNA and pLJ85 for TR-DNA); Lane 3: negative control (genomic DNA from non-transformed root); Lane 4-13: genomic DNA of Ri-transformed root lines A413, A414, A416, A417, A418, A419, A420, A422, A426, and A428, respectively. Supplementary material 1 (TIFF 9619 kb)

10725_2014_5_MOESM2_ESM.tif

Fig. S2 Expression of rol genes at transcription level in Ri-transformed roots observed by RT-PCR using rolA-, rolB-, rolC- and rolD- specific primers. Lane 1: molecular marker (100 bp DNA ladder, 100 bp DNA ladder for rolA and rolC); Lane 2: positive control (pLJ1, containing TL-DNA); Lane 3: negative control (Genomic DNA of non-transformed root); Lane 4-11: amplified cDNAs of Ri-transformed root lines A414, A416, A417, A418, A419, A420, A422, A426, and A428, respectively. Supplementary material 2 (TIFF 829 kb)

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Roychowdhury, D., Basu, A. & Jha, S. Morphological and molecular variation in Ri-transformed root lines are stable in long term cultures of Tylophora indica . Plant Growth Regul 75, 443–453 (2015). https://doi.org/10.1007/s10725-014-0005-y

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