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In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

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Abstract

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l−1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l−1 each of Kin, BA and 0.1 mg l−1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite®, if their base was treated with 500 mg l−1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M5 medium having 0.5 mg l−1 each of 2, 4-D and Kin, 100 mg l−1 l-phenylalanine and 5 mg l−1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.

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Abbreviations

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

BA:

6-Benzyladenine

IBA:

Indole-3-butyric acid

Kin:

Kinetin

MS:

Murashige and Skoog (1962) medium

NAA:

α-Naphthalene acetic acid

PFD:

Photon flux density

PGRs:

Plant growth regulators

RH:

Relative humidity

TLC:

Thin layer chromatography

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Acknowledgments

Authors (DL and AKP) gratefully acknowledge the financial support from Council of Scientific and Industrial Research (CSIR), New Delhi in the form of Junior and Senior Research Fellowship. We are thankful to Dr. Mahendra Bhati, Department of Chemistry, Jai Narain Vyas University, Jodhpur (India) for his duly support in determination of MP of obtained crystals. Authors are also thankful to Dr. Kamini Dinesh (Professor of English) for the language improvement of the present manuscript.

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Authors and Affiliations

  1. Biotechnology Unit, Department of Botany (UGC-Centre of Advanced Study), Jai Narain Vyas University, New Campus, Prof. V.V. John Road, Jodhpur, 342 001, Rajasthan, India

    Deepika Lodha, Ashok Kumar Patel, Manoj K. Rai & N. S. Shekhawat

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  1. Deepika Lodha
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  2. Ashok Kumar Patel
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  3. Manoj K. Rai
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  4. N. S. Shekhawat
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Corresponding author

Correspondence to Deepika Lodha.

Additional information

Communicated by M. H. Walter.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1 (ESM_1)

Figures showing extraction and confirmation of alkaloid contents from callus culture of E. foliata. Supplementary material 1 (PDF 261 kb)

Online Resource 2 (ESM_2)

Table showing identification of product extracts of E. foliata by TLC. Supplementary material 2 (PDF 123 kb)

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Lodha, D., Patel, A.K., Rai, M.K. et al. In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs. Acta Physiol Plant 36, 3071–3079 (2014). https://doi.org/10.1007/s11738-014-1677-7

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