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Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

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Abstract

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L−1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L−1 BAP and 0.4 mg L−1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L−1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.

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Abbreviations

BAP:

6-Benzylaminopurine

Cerpegin:

1,1-Dimethylfuro(3,4-C)pyridine-3,4(1H, 5H)-dione

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

DSO:

Direct shoot organogenesis

FW:

Fresh weight

IAA:

Indole 3-acetic acid

IBA:

Indole 3-butyric acid

2iP:

6-γ,γ-Dimethylallylaminopurine

ISO:

Indirect shoot organogenesis

ISSR:

Inter simple sequence repeats

MS:

Murashige and Skoog’s medium (1962)

NAA:

α-Naphthalene acetic acid

PGRs:

Plant growth regulators

PR:

Picloram (4-amino-3,5,6-trichloropicolinic acid)

RAPD:

Random amplified polymorphic DNA

RE:

Rutin equivalent

RP-HPLC:

Reverse phase-high performance liquid chromatography

TAE:

Tannic acid equivalent

TPC:

Total phenolic content

TFC:

Total flavonoid content

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Acknowledgments

Junior research fellowship (JRF) to JJC and financial support for this work from Department of Biotechnology (DBT), Govertment of India, New Delhi is gratefully acknowledged. Authors are also thankful to Dr. V. A. Bapat, Emeritus Scientist from Plant cell culture technology section, Nuclear agriculture and biotechnology division, Bhabha Atomic Research Centre (BARC) Mumbai, India for his constructive comments on the manuscript.

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Authors and Affiliations

  1. Department of Botany, Shivaji University, Kolhapur, 416 004, Maharashtra, India

    J. J. Chavan, N. B. Gaikwad, S. D. Umdale, P. R. Kshirsagar & S. R. Yadav

  2. National Research Centre on DNA Fingerprinting, NBPGR, New Delhi, 110 012, India

    S. D. Umdale & K. V. Bhat

  3. Department of Botany, Yashavantrao Chavan Institute of Science, Satara, 415 001, Maharashtra, India

    J. J. Chavan

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  1. J. J. Chavan
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  2. N. B. Gaikwad
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Chavan, J.J., Gaikwad, N.B., Umdale, S.D. et al. Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui . Plant Growth Regul 72, 1–15 (2014). https://doi.org/10.1007/s10725-013-9830-7

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