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Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

Applied Biochemistry and Biotechnology volume 177, pages 118–136 (2015)Cite this article

Abstract

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

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Abbreviations

BA:

6-Benzyladenine

IBA:

Indole-3-butyric acid

Kin:

Kinetin

NAA:

α Naphthalene acetic acid

MS:

Murashige and Skoog’s medium

2-iP:

2-Isopentenyl adenine

NS:

Nodal segment

RAPD:

Random amplification polymorphic DNA

DAMD:

Direct amplification of minisatellite DNA

SPAR:

Single primer amplification reaction

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Acknowledgments

Research support provided by Dr. D. S. Kothari Postdoctoral Fellowship, University Grants Commission (UGC), Govt. of India, New Delhi, is duly acknowledged.

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Affiliations

  1. Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, 202 002, India

    Nigar Fatima & Iqbal Ahmad

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

    Naseem Ahmad & Mohammad Anis

  3. Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia

    Mohammad Anis

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  1. Nigar Fatima
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Fatima, N., Ahmad, N., Ahmad, I. et al. Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.. Appl Biochem Biotechnol 177, 118–136 (2015). https://doi.org/10.1007/s12010-015-1732-x

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Keywords

  • Plant growth regulators
  • Axillary bud proliferation
  • Somaclonal variations
  • Genetic stability
  • Micropropagation