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Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas

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Abstract

Nardostachys jatamansi (D. Don) DC., a small, perennial, rhizomatous herb of immense medicinal importance since ancient times, is restricted to specialized habitats of alpine Himalayas ranging from 3000 to 5200 m asl. The species has been recently listed as critically endangered under IUCN Red list of threatened species due to over exploitation of its rhizomes for medicinal uses, habitat degradation, trade and other biotic and anthropogenic interferences. An efficient protocol using both indirect and direct shoot organogenesis has been optimized for N. jatamansi. Best callusing was achieved from the cut ends of leaf and petiole explants within 15 days of culture in MS medium supplemented with 1.5 mg/l α-naphthalene acetic acid and 1.0 mg/l meta-Topolin. Culturing the explants at low temperature (13 ± 1 °C) resulted in better callus growth, shoot regeneration, hyperhydricity control and improvement in photosynthetic pigment content in regenerated shoots. Also, direct organogenesis from shoot tip and petiole explants was achieved in MS medium containing 1.0 mg/l meta-Topolin. Optimum rooting was achieved in the same medium supplemented with 1.0 mg/l indole acetic acid wherein averages of 4.52 roots/shoot were induced. Genetic stability of in vitro-derived plantlets was assessed and compared to mother plant using molecular markers and flow cytometry. Intron Splice Junction (ISJ) and Start Codon Targeted polymorphism (SCoT) marker based profiling revealed uniform banding profile in case of direct shoot organogenesis (DSO)-derived plants while callus mediated organogenesis (CMO)-derived plants showed slight variations as compared to mother plant. The genome size of N. jatamansi was found to be 2C = 1.40 ± 0.01 pg and therefore 684.6 Mbp (1C). Although organogenic calli showed mixoploidy but no major phenotypic and genetic rearrangements were detected by flow cytometry in callus-derived plants. Significantly higher antioxidant activity was observed in callus-derived plants as compared to mother and DSO-derived plants. Plant parts, regeneration pathways and various solvent systems greatly affected the yields of total phenolics, flavonoids, alkaloids, tannins contents present in the in vitro raised plantlets.

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Abbreviations

SCoT:

Start Codon Targeted polymorphism

ISJ:

Intron Splice Junction

FCM:

Flow cytometry

LB01:

Lysis buffer 01

MS medium:

Murasshige and Skoog medium (1962)

NAA:

α-naphthaleneacetic acid

2-4D :

2,4-dichlorophenoxyacetic acid

mT:

Meta-Topolin

KN:

Kinetin

2-iP:

N6–[2-isopentenyl] adenine

BAP:

6-benzylaminopurine

TDZ:

Thidiazuron

IAA:

Indole-3 acetic acid

IBA:

Indole-3-butyric acid

PGRs:

Plant growth regulators

DSO:

Direct shoot organogenesis

CMO:

Callus mediated organogenesis

PCR:

Polymerase chain reaction

Mbp:

Mega base pairs

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Acknowledgments

Financial supports received from DBT research grant no. BT/PR-10533/BCE/08/622/2008 and other DBT funded projects are gratefully acknowledged. The authors would like to thank the Bioinformatics Centre, NEHU and Prof. A. Chatterjee, Dr. N. Ghosal from Department of Biotechnology and Bioinformatics, NEHU for providing the facilities carried out during the course of study.

Authors’ contributions statement

BB carried out the experiments, analyzed the data and drafted the manuscript. All the authors were involved in the designing of the experiments. SK and PT supervised the work. SK edited the manuscript. All authors read and approved the final version of this manuscript.

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  1. Plant Biotechnology Laboratory, Department of Botany, North-Eastern Hill University, Shillong, 793022, Meghalaya, India

    Biswajit Bose, Suman Kumaria, Hiranjit Choudhury & Pramod Tandon

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  1. Biswajit Bose
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Bose, B., Kumaria, S., Choudhury, H. et al. Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas. Plant Cell Tiss Organ Cult 124, 331–349 (2016). https://doi.org/10.1007/s11240-015-0897-x

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