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Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid

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Abstract

Malaxis wallichii (Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200–2000 m asl. The pseudobulbs are important ingredients of century old drug ‘Ashtavarga’ and a polyherbal energetic tonic ‘Chyavanprash’. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for M. wallichii. In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5 ± 0.6) was observed in MS medium supplemented with 1.0 mg/l meta-topolin and 0.5 mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66 ± 0.3) was achieved in half-strength MS medium fortified with 1.0 mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of M. wallichii (2n) was found to be 2C = 2.760 ± 0.02 pg and therefore, 1349.64 Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased H2O2 content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

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Acknowledgements

Financial support received from Department of Biotechnology (DBT), Government of India, New Delhi vide research Grant No. BT/PR-10533/BCE/08/622/2008 is gratefully acknowledged. The authors would like to thank Prof. Anupam Chatterjee, Dr. Nitin Ghosal from Department of Biotechnology and Bioinformatics, NEHU for FCM instrumentation facility.

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  1. Pramod Tandon

    Present address: Biotech Park, Lucknow, Uttar Pradesh, 226021, India

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

    Biswajit Bose, Suman Kumaria & Pramod Tandon

  2. Biotechnology Laboratory, Unit of Biotechnology, Department of Basic Sciences and Social Sciences, School of Technology, North-Eastern Hill University, Shillong, Meghalaya, 793022, India

    Hiranjit Choudhury

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

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Bose, B., Kumaria, S., Choudhury, H. et al. Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid. Physiol Mol Biol Plants 23, 955–968 (2017). https://doi.org/10.1007/s12298-017-0474-3

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