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Embryogenesis followed by enhanced micro-multiplication and eco-restoration of Calamus thwaitesii Becc.: an economic non-wood forest produce for strengthening agroforestry system

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Abstract

The present study is focussed on development of a high-frequency micro-multiplication system in Calamus thwaitesii, through somatic embryogenesis from immature zygotic embryos cultured in Murashige and Skoog (MS) medium supplemented with 31.68 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The semi-friable calli when cultured in the same medium augmented with 2.22 µM 6-benzylaminopurine (BAP) and 1.07 µM α-naphthalene acetic acid (NAA) induced ~ 12 discrete globular embryoids in 6 weeks. The isolated embryoids in hormone-free media yielded 65% plantlets. Furthermore, embryoids and axenic shoots exhibited maximum shoot induction in medium supplemented with 0.45 µM Thidiazuron (TDZ). The shoot initials after subculture in media supplemented with 1.78 µM BAP and 0.45 µM TDZ produced shoot proliferation followed by elongation in basal medium. The elongated shoots produced roots in media supplemented with 16.11 µM NAA. With this established protocol, ~ 5940 rooted plantlets could be harvested after 40 weeks from a single embryoid. Genetic stability analysis of the plantlets using inter simple sequence repeat markers recorded only 0.05% genetic polymorphism. The plantlets were hardened in a mist house for 8 weeks, and then to 50% shade house for another 16 weeks, and the well-established 6-month-old nursery plants, reintroduced to selected forest segments, exhibited 86% field establishment even after 3 years of observation. Thus, the mass multiplication system developed could be a breakthrough for large-scale multiplication of C. thwaitesii to ensure continuous supply of quality planting material to the cottage industry through the development of agroforestry systems. Furthermore, the in vitro culture system developed here can be replicated for research activities related to the long-term–short-term conservation, micro-multiplication and sustainable utilization of rare, endangered, endemic, monopodial/single stemmed rattan palms.

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Notes

  1. Annealing temperature of the primers ranges from 50 to 55 °C for the different primers used in this study.

Abbreviations

BAP:

N6-benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

IBA:

Indole-3-butyric acid

ISSR:

Inter simple sequence repeat

MS:

Murashige and Skoog

NAA:

α-Naphthaleneacetic acid

PGR:

Plant growth regulator

TDZ:

Thidiazuron

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Acknowledgements

We thankfully acknowledge the Department of Biotechnology (DBT), Government of India, for providing the financial assistance through the research grant (No BT/R&D/08/04/95), the Director, Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI) for providing laboratory facilities and the Department of Forests, Government of Kerala extended support for eco-restoration activities.

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Authors and Affiliations

  1. Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, 695 562, India

    Achuthan Sudarsanan Hemanthakumar

  2. Department of Botany, University College, Thiruvananthapuram, Kerala, India

    Thankappan Suvarna Preetha

  3. Department of Genomic Science, Central University of Kerala, Kasaragod, Kerala, India

    Padmesh Pandaram Pillai

  4. School of Biosciences, Mar Athanasios College for Advanced Studies, Tiruvalla, Kerala, India

    Sooriamuthu Seeni

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  1. Achuthan Sudarsanan Hemanthakumar
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Correspondence to Thankappan Suvarna Preetha.

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Hemanthakumar, A.S., Preetha, T.S., Pillai, P.P. et al. Embryogenesis followed by enhanced micro-multiplication and eco-restoration of Calamus thwaitesii Becc.: an economic non-wood forest produce for strengthening agroforestry system. Agroforest Syst 93, 1093–1105 (2019). https://doi.org/10.1007/s10457-018-0207-9

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