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Ex Situ Conservation of Phyllanthus fraternus Webster and Evaluation of Genetic Fidelity in Regenerates Using DNA-Based Molecular Marker

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Abstract

Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5 ± 2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.

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Abbreviations

BAP:

6-Benzylaminopurine

NAA:

α-Naphthaleneacetic acid

MS:

Murashige and Skoog

RAPD:

Random amplified polymorphic DNA

ISSR:

Inter-simple sequence repeat analysis

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Acknowledgments

Richa Upadhyay is highly thankful to CSIR, New Delhi, for providing fellowship in the form of SRF.

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Authors and Affiliations

  1. Department of Botany, MMV, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India

    Richa Upadhyay, Chandra Shekhar Singh & Kavindra Nath Tiwari

  2. Crop Improvement Division, Indian Institute of Vegetable Research, Shanahshahpur, Varanasi, Uttar Pradesh, 221305, India

    Sarvesh Pratap Kashyap & Major Singh

  3. Department of Zoology, MMV, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India

    Karuna Singh

Authors
  1. Richa Upadhyay
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  2. Sarvesh Pratap Kashyap
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  3. Chandra Shekhar Singh
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  4. Kavindra Nath Tiwari
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  5. Karuna Singh
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  6. Major Singh
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Correspondence to Kavindra Nath Tiwari.

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Upadhyay, R., Kashyap, S.P., Singh, C.S. et al. Ex Situ Conservation of Phyllanthus fraternus Webster and Evaluation of Genetic Fidelity in Regenerates Using DNA-Based Molecular Marker. Appl Biochem Biotechnol 174, 2195–2208 (2014). https://doi.org/10.1007/s12010-014-1175-9

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  • DOI: https://doi.org/10.1007/s12010-014-1175-9

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