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An Efficient In Vitro Process for Cyclic Clonal Production of Shoots from Adult Tree of Cassia alata L. and Evaluation of Genetic Stability Using DNA-Based Markers

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Abstract

An efficient, cyclic, two-step protocol for clonal in vitro regeneration system of an antiallergenic plant, Cassia alata, has been successfully developed. Nodal explants from a 5-year-old tree were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 μM) of thidiazuron (TDZ). TDZ (5.0 μM) was found to be optimal for the formation of maximum shoot induction. Shoot proliferation and elongation increased when the regenerated shoots were subcultured on hormone-free MS medium after 4 weeks of exposure to TDZ. Nodal explants from in vitro regenerated microshoots to developed shoots, thus making the process recurrent. In 6 months duration, owing to the recurring nature of the protocol, large number of shoots could be produced from a single nodal explant from an adult tree. Shoots rooted best on MS supplemented medium with 0.5 μM IBA. Regenerated plantlets were acclimatized and successfully transplanted to the garden soil, where they grew well without any morphological and genetic variations. To confirm the uniformity, the genetic fidelity of in vitro raised C. alata clones was also assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The present regeneration process not only favored the clonal multiplication but also expressed the regeneration capability of in vitro regenerated microshoots and can be subjugated for catering enough raw materials to various pharma industries by continuous cyclic shoot production.

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Acknowledgments

Md. Rafique Ahmed is thankful to the University Grants Commission (UGC), Goverment of India, New Delhi for the award of Senior Research Fellow (MANF/UGC). Research support provided by the Department of Science and Technology and University Grants Commission under DST-FIST (2011–16) and UGC-SAP, DRS-I (2009–2014) Programme respectively is duly acknowledged.

Contributions

MR Ahmed designed and performed the whole experiment and performed genetic integrity experiments. Hashim A. Al-Etta analyzed the statistical data. The whole experiment was guided by Prof. M. Anis who also edited the manuscript.

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Authors and Affiliations

  1. Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, 202 002, India

    Md Rafique Ahmed & Mohammad Anis

  2. Department of Plant Production, College of Food & Agricultural Sciences, King Saud University, PO Box 2460, Riyadh, 11451, Saudi Arabia

    Mohammad Anis & Hashim A. Al-Etta

Authors
  1. Md Rafique Ahmed
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  2. Mohammad Anis
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  3. Hashim A. Al-Etta
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Correspondence to Mohammad Anis.

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Ahmed, M.R., Anis, M. & Al-Etta, H.A. An Efficient In Vitro Process for Cyclic Clonal Production of Shoots from Adult Tree of Cassia alata L. and Evaluation of Genetic Stability Using DNA-Based Markers. Appl Biochem Biotechnol 174, 2886–2896 (2014). https://doi.org/10.1007/s12010-014-1234-2

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  • DOI: https://doi.org/10.1007/s12010-014-1234-2

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