Acta Physiologiae Plantarum

, Volume 35, Issue 3, pp 791–800 | Cite as

Change in total phenolic content and antibacterial activity in regenerants of Vitex negundo L.

  • Naseem AhmadEmail author
  • Md Imran Khan
  • Sarfaraz Ahmed
  • Saad Bin Javed
  • Mohammad Faisal
  • Mohammad Anis
  • Sumbul Rehman
  • Syed Mohammad Umair
Original Paper


A valuable medicinal plant, Vitex negundo L. has been investigated for its regeneration potential using shoot tip explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine, 2-isopentenyl adenine] used as supplement to Murashige and Skoog medium (MS), BA at 5.0 μM concentration proved best for multiple shoot induction yielding 3.60 ± 0.50 shoots after 8 weeks of culture. Inclusion of a low concentration of an auxin with optimal cytokinin concentration favoured shoot multiplication and the optimum response was observed on MS medium supplemented with BA (5.0 μM) along with α Naphthalene acetic acid (0.5 μM), where 65.0 ± 1.73 % cultures responded with a mean number of 4.80 ± 0.58 shoots per explants after 8 weeks of culture. Ex vitro rooting of in vitro derived microshoots was achieved upon dipping the cut ends of microshoots in 500 μM indole-3-butyric acid for 10 min followed by transfer to thermocol cups containing sterile soilrite. About 95 % of the plantlets survived the acclimatization procedure and were transferred to greenhouse and finally to field. Screening of the antibacterial activity and estimation of total phenolic content of ethanolic extracts of micropropagated plants were also carried out and compared with that of the mother plant.


Vitex negundo L. Regeneration Acclimatization Antimicrobial activity Total phenolic content 



6-Benzyl adenine


Indole-3-acetic acid


Indole-3-butyric acid


2-Isopentenyl adenine




Plant growth regulators


Murashige and Skoog (1962) medium


α Naphthalene acetic acid



The award of DST, Young Scientist (SR/FT/LS-014/2009) Scheme to Naseem Ahmad by the Department of Science and Technology (DST), Government of India, New Delhi, is greatly acknowledged. Research support from the Department of Science and Technology (Govt. of India) New Delhi under the DST-FIST (2011) and UGC-SAP (2009) Programme, is also acknowledged.


  1. Afroz F, Hassan AKM, Bari LS, Sultana R, Munshi JL, Jahan MAA, Khatun R (2008) In vitro regeneration of Vitex negundo L., a woody valuable medicinal plant through high frequency axillary shoot proliferation. Bangladesh J Sci Ind Res 43:345–352CrossRefGoogle Scholar
  2. Ahmad N, Anis M (2007a) Rapid clonal multiplication of a woody tree, Vitex negundo L. through axillary shoots proliferation. Agrofor Syst 71:195–200CrossRefGoogle Scholar
  3. Ahmad N, Anis M (2007b) Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.). J Hortic Sci Biotech 82:585–589Google Scholar
  4. Ahmad N, Anis M (2011) An efficient in vitro process for recurrent production of cloned plants of Vitex negundo L. Eur J For Res 130:135–144CrossRefGoogle Scholar
  5. Ahmad N, Wali SA, Anis M (2008) In vitro production of true-to-type plants of Vitex negundo L. from nodal explants. J Hortic Sci Biotech 83:313–317Google Scholar
  6. Anis M, Husain MK, Faisal M, Shahzad A, Ahmad N, Siddique I, Khan H (2009) In vitro approaches for plant regeneration and conservation of some medicinal plants. In: Kumar A, Sopory SK (eds) Recent advances in plant biotechnology and its applications. IK International Pvt Ltd, New Delhi, pp 397–410Google Scholar
  7. Anonymous (2003) The wealth of India, raw material, vol 1, Publication and Information Directorate, CSIR, New Delhi p 522–524Google Scholar
  8. Balaraju K, Agastian P, Preetamraj JP, Asokiyaraj S, Ignacimuthu S (2008) Micropropagation of Vitex agnus-castus (Verbenaceae) a valuable medicinal plant. In Vitro Cell Dev Biol Plant 44:436–441CrossRefGoogle Scholar
  9. Chandramu C, Manohar RD, Krupadanam DGL, Dashavantha RV (2003a) Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L. Phytother Res 17:129–134PubMedCrossRefGoogle Scholar
  10. Chandramu C, Rao DM, Reddy VD (2003b) High frequency induction of multiple shoots from nodal explants of Vitex negundo L. using sodium sulphate. J Plant Biotech 5:107–113Google Scholar
  11. De klerk GJ (2002) Rooting of micro-cuttings: theory and practice. In Vitro Cell Dev Biol Plant 38:415–422CrossRefGoogle Scholar
  12. Esekhiagbe M, Agatemor MMU, Agatemor C (2009) Phenolic content and antimicrobial potentials of Xylopia aethiopica and Myristica argentea. Maced J Chem Chem Eng 28:159–162Google Scholar
  13. Faisal M, Anis M (2002) Rapid in vitro propagation of Rauvolfia tetraphylla L.—an endangered medicinal plant. Physiol Mol Biol Plant 8:295–299Google Scholar
  14. Faisal M, Ahmad N, Anis M (2005) Shoot multiplication in Rauvolfia tetraphylla using thidiazuron. Plant Cell Tissue Organ Cult 80:187–190CrossRefGoogle Scholar
  15. Faisal M, Siddiqui I, Anis M (2006) In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens—a valuable medicinal plant. Ann App Biol 148:1–6CrossRefGoogle Scholar
  16. Hembrom ME, Martin KP, Patchathundikandi SK, Madassery J (2006) Rapid in vitro production of true-to-type plants of Pogostemon heyneanus through dedifferentiated axillary buds. In Vitro Cell Dev Biol Plant 42:283–286CrossRefGoogle Scholar
  17. Khan MI, Ahmad N, Anis M (2011) The role of cytokinins on in vitro shoot production in Salix tetrasperma Roxb.: a tree of ecological importance. Trees 25:577–584CrossRefGoogle Scholar
  18. Lin YL, Juan IM, Chen YL, Liang YC, Lin JK (1999) Composition of polyphenols in fresh tea leaves and associations of their oxygen-radical-absorbing capacity with antiproliferative actions in fibroblast cells. J Agric Food Chem 44:1387–1394CrossRefGoogle Scholar
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–494CrossRefGoogle Scholar
  20. NCCLS (1995) Method comparison and bias estimation using patient samples. Approved guideline. NCCLS document EP9-A. NCCLS, WayneGoogle Scholar
  21. Pandjaitan N, Howard LK, Morelock T, Cril MI (2005) Antioxidant capacity and phenolic content of spinach as affected by genetics and maturation. J Agric Food Chem 53:8618–8623PubMedCrossRefGoogle Scholar
  22. Preece JE, Shutter EG (1991) Acclimatization of micropropagated plants to the greenhouse and field. In: Debergh PC, Zimmerman RH (eds) Micropropagation technology and application. Kluwer, Dordrecht, pp 71–93CrossRefGoogle Scholar
  23. Rout GR, Mallik UC, Das P (2000) In vitro manipulation and of Plumbago zeylanica Linn. Plant Growth Regul 28:1–4CrossRefGoogle Scholar
  24. Sahoo Y, Chand PK (1998) Micropropagation of Vitex negundo L. a woody aromatic medicinal shrub, through high frequency axillary shoot proliferation. Plant Cell Rep 18:301–307CrossRefGoogle Scholar
  25. Sharma N, Chandel KPS, Paul A (1993) In vitro propagation of Gentiana kurroo: an indigenous threatened plant of medicinal importance. Plant Cell Tissue Organ Cult 34:307–309CrossRefGoogle Scholar
  26. Shasany AK, Khanuja SPS, Dhawan S, Yadav U, Sharma S, Kumar S (1988) High regenerative nature of Mentha arvensis internodes. J Biosci 23:641–646CrossRefGoogle Scholar
  27. Siddique I, Anis M (2009) Direct plant regeneration from nodal explants of Balanites aegyptiaca L. (Del.)—a valuable medicinal tree. New For 37:53–62CrossRefGoogle Scholar
  28. Sreelatha S, Padma PR (2009) Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr 64:303–311PubMedCrossRefGoogle Scholar
  29. Swain T, Hillis E (1959) The phenolic constituents of Prunus domestica I. The quantitative analysis of phenolic constituents. J Sci Food Agric 10:63–68CrossRefGoogle Scholar
  30. Thiruvengadam M, Jayabalan N (2011) In vitro flowering of Vitex negundo L., a medicinal plant. Plant Cell Biotech Mol Biol 2:67–70Google Scholar
  31. Tsay HS, Gau TG, Chen CC (1989) Rapid clonal propagation of Pinellia ternate by tissue culture. Plant Cell Rep 8:450–454CrossRefGoogle Scholar
  32. Usha PK, Benjamin S, Mohanan KV, Raghu AV (2007) An efficient micropropagatin system for Vitex negundo L., an important woody aromatic medicinal plant, through shoot tip culture. Res J Bot 2:102–107CrossRefGoogle Scholar
  33. Vadawale AV, Barve DM, Dave AM (2006) In vitro flowering and rapid propagation of Vitex negundo L.—a medicinal plant. Indian J Biotech 5:112–116Google Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012

Authors and Affiliations

  • Naseem Ahmad
    • 1
    Email author
  • Md Imran Khan
    • 1
  • Sarfaraz Ahmed
    • 2
  • Saad Bin Javed
    • 1
  • Mohammad Faisal
    • 3
  • Mohammad Anis
    • 1
  • Sumbul Rehman
    • 4
  • Syed Mohammad Umair
    • 4
  1. 1.Plant Biotechnology Laboratory, Department of BotanyAligarh Muslim UniversityAligarhIndia
  2. 2.Natural Product Laboratory, Department of ChemistryAligarh Muslim UniversityAligarhIndia
  3. 3.Department of Botany and Microbiology, College of ScienceKing Saud UniversityRiyadhKingdom of Saudi Arabia
  4. 4.Department of Ilmul Advia, Faculty of Unani MedicineAligarh Muslim UniversityAligarhIndia

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