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Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid

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Abstract

An efficient in vitro plant regeneration system was established through somatic embryogenesis for Anoectochilus elatus Lindley, an endangered jewel orchid. Direct somatic embryogenesis was achieved from nodal explants (17.4 embryos per explant with 63.4% response) on Mitra medium supplemented with Morel vitamins, thidiazuron (4.54 µM) and ∞-naphthaleneacetic acid (2.69 µM). Simultaneously, a protocol was developed for indirect somatic embryogenesis from internodal explant, produced embryogenic calli and embryos (31.3 embryos with 76.4% response) on same medium amended with 50 mg/L peptone and 5% coconut water. Both types of embryogenic pathways, produced morphologically similar globular embryos in the form of protocorm like bodies and successfully germinated on hormone free Mitra medium supplemented with Morel vitamins. Morpho-histological investigation of the embryo revealed the initiation and developmental features of somatic embryos. In vitro regenerated plantlets were successfully established from heterotrophic to a photoautotrophic stage by reducing the nutrient content in culture media, adjusting temperature and humidity through three step method. During the process, no morphological and physiological abnormalities were observed. Hardened plantlets were successfully acclimatized at poly tunnel chamber with 95% of survival rate. Further, inter simple sequence repeats (ISSRs) molecular markers were used to analyse the genetic homogeneity of regenerated plants. Analysis with this method showed that the homogeneity is comparatively higher in direct somatic embryo regenerated plants (94.22%) as compared to plants elevated from an indirect somatic embryo (93.05%). The present study provides morpho-histological and genetically stable plants for germplasm conservation and further utility of this endangered jewel orchid.

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Abbreviations

2,4-D:

2,4-diclorophenoxyacetic acid

2iP:

N6-(2-isopentenyl) adenine

BA:

N6-benzyleadenine

CW:

Coconut water

DSE:

Direct somatic embryogenesis

IDSE:

Indirect somatic embryogenesis

ISSRs:

Inter simple sequence repeats

NAA:

∞-naphthaleneacetic acid

PEP:

Peptone

PGRs:

Plant growth regulators

PLBs:

Protocorm like bodies

SEM:

Scanning electron microscopy

TDZ:

Thidiazuron

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Acknowledgements

The corresponding author is grateful to the University Grant Commission (UGC), Delhi, India for providing Emeritus Fellowship. All the authors are grateful to Dr. P. Palani, Assistant Professor, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India, for valuable help in scanning electron microscopic observation.

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Authors and Affiliations

  1. PG & Research Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, 620020, India

    N. Ahamed Sherif

  2. Southern Regional Centre, Botanical Survey of India, TNAU Campus, Coimbatore, Tamil Nadu, 641003, India

    J. H. Franklin Benjamin

  3. Department of Botany, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India

    N. Ahamed Sherif, T. Senthil Kumar & M. V. Rao

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  1. N. Ahamed Sherif
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  2. J. H. Franklin Benjamin
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  3. T. Senthil Kumar
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  4. M. V. Rao
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NAS, JHFB, TSK and MVR are equally contributed to bringing out the manuscript in a successful manner.

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Correspondence to M. V. Rao.

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Communicated by Sergio J. Ochatt.

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Sherif, N.A., Franklin Benjamin, J.H., Senthil Kumar, T. et al. Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid. Plant Cell Tiss Organ Cult 132, 303–316 (2018). https://doi.org/10.1007/s11240-017-1330-4

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