Applied Biochemistry and Biotechnology

, Volume 177, Issue 1, pp 118–136 | Cite as

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.

  • Nigar FatimaEmail author
  • Naseem Ahmad
  • Iqbal Ahmad
  • Mohammad Anis


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.


Plant growth regulators Axillary bud proliferation Somaclonal variations Genetic stability Micropropagation 





Indole-3-butyric acid




α Naphthalene acetic acid


Murashige and Skoog’s medium


2-Isopentenyl adenine


Nodal segment


Random amplification polymorphic DNA


Direct amplification of minisatellite DNA


Single primer amplification reaction



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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nigar Fatima
    • 1
    Email author
  • Naseem Ahmad
    • 2
  • Iqbal Ahmad
    • 1
  • Mohammad Anis
    • 2
    • 3
  1. 1.Department of Agricultural MicrobiologyAligarh Muslim UniversityAligarhIndia
  2. 2.Plant Biotechnology Laboratory, Department of BotanyAligarh Muslim UniversityAligarhIndia
  3. 3.Department of Plant Production, College of Food and Agriculture SciencesKing Saud UniversityRiyadhSaudi Arabia

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