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Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC

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Abstract

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.

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Abbreviations

CTAB:

Cetyl trimethyl ammonium bromide

DMRT:

Duncan’s multiple range test

f.w.:

Fresh weight

HPLC:

High performance liquid chromatography

ISSR:

Inter simple sequence repeat

MS:

Murashige and Skoog

PDA:

Photo Diode Array

RAPD:

Random amplified polymorphic DNA

SPSS:

Statistical package for social sciences

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Acknowledgments

V. Agrawal is grateful to the University Grants Commission, New Delhi for providing financial assistance in the form of a UGC’s Major Research Project and to the University of Delhi, Delhi for sanctioning funds for Research and Development. SKC is indebted to Indian Council of Medical Research, New Delhi for the award of SRF.

Conflict of interest

The authors declare that they have no conflict of interest.

Author contributions statement

This paper pertains to direct regeneration through mature in vivo nodal explants of Cassia angustifolia and establishment of genetic and biochemical uniformity amongst the regenerants employing RAPD and HPLC, respectively. All the authors have equal contribution for the research paper.

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Authors and Affiliations

  1. Department of Botany, University of Delhi, Delhi, 110007, India

    Siva K. Chetri, Pratima Rani Sardar & Veena Agrawal

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  1. Siva K. Chetri
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  2. Pratima Rani Sardar
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  3. Veena Agrawal
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Correspondence to Veena Agrawal.

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Chetri, S.K., Sardar, P.R. & Agrawal, V. Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC. Physiol Mol Biol Plants 20, 517–526 (2014). https://doi.org/10.1007/s12298-014-0257-z

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  • DOI: https://doi.org/10.1007/s12298-014-0257-z

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