Applied Biochemistry and Biotechnology

, Volume 169, Issue 7, pp 2004–2015 | Cite as

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

  • Nisar Ahmad
  • Bilal Haider Abbasi
  • Inayat ur Rahman
  • Hina Fazal


A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L−1 6-benzyladenine (BA) along with 0.5 mg L−1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L−1 BA in combination with 0.25 mg L−1 α-naphthalene acetic acid (NAA) and 0.25 mg L−1 2,4-dichlorophenoxyacetic acid or 0.5 mg L−1 indole butyric acid (IBA) along with 0.25 mg L−1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L−1 thiodiazoran or 1.5 mg L−1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.


Piper nigrum L. In vitro regeneration Antioxidant enzyme activities HPLC Piperine 







Gibberellic acid


α-Naphthalene acetic acid


2,4-Dichlorophenoxyacetic acid


Indole butyric acid


Indole acetic acid


MS medium without plant growth regulators


Plant growth regulators


Superoxide dismutase






Ascorbate peroxidase


World Health Organization


Relative standard deviation



Financial support of Higher Education Commission of Pakistan is acknowledged.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Nisar Ahmad
    • 1
  • Bilal Haider Abbasi
    • 1
  • Inayat ur Rahman
    • 2
  • Hina Fazal
    • 2
  1. 1.Department of Biotechnology, Faculty of Biological SciencesQuaid-i-Azam UniversityIslamabadPakistan
  2. 2.Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories ComplexPeshawarPakistan

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