Optimization of a Liquid Culture System for Shoot Regeneration and Achieving an Enriched Level of Scopadulcic Acid B in the Leaf Organ Cultures of Scoparia dulcis L. by Response Surface Methodology

  • Gandhi PremkumarEmail author
  • Thirupathi KaruppanapandianEmail author
  • Chandran Sureshpandian
  • Neelakanda Arumugam
  • Avadayappan Selvam
  • Kaniappan Rajarathinam
Plant Development/Regeneration


Response surface methodology (RSM) approach was utilized in the present investigation to optimize the constituents of Murashige and Skoog’s (MS) liquid medium to enrich the scopadulcic acid B (SDB) content in Scoparia dulcis L. RSM approach using the central composite design (CCD) was employed to identify the precise concentration of growth regulators of medium and substantiated in shake-flask cultivation. CCD-RSM model revealed a strong agreement, with a predicted coefficient of determination (R2) values of 0.881 and 0.872 for the shoot regeneration percentage (Y1) and the average number of shoots per explant (Y2), respectively. RSM predicted augmented conditions of MS liquid medium considerably influence the shoot proliferation (Y1 and Y2) in the leaf explants of S. duclis. The medium was fortified with 3.59 μM kinetin (KN; X1), 6.00 μM 6-benzylaminopurine (BAP; X2), 3.93 μM indole-3-acetic acid (IAA; X3), and 25.84 g L−1 of sucrose (X4). The present investigation conquered a maximal response of Y1 and Y2 with 91.28 ± 3.85 and 82.26 ± 2.13, respectively. The experimentally detected values are in close agreement with the predicted values of 90.07 and 79.70, respectively. This proposed that the developed design using CCD had efficacy in the optimization of medium components. The enhanced level of SDB, 9.89 ± 0.98 mg g−1 FW (ca. 5-fold), in the plantlets grown on liquid medium was significantly greater than those accomplished in the other tested plant tissues, comprises of field-grown parent plant leaves and in vitro developed callus and micropropagated plants on MS solid medium. For the first time, the methodology was developed effectively using RSM to promote the shoot proliferation and enhance the SDB production in the leaf organ culture of S. dulcis on MS liquid medium.


CCD-RSM model Growth regulators Murashige and Skoog’s medium Scoparia dulcis Shake-flask culture 



The authors are grateful to Prof. Russell V. Lenth, Department of Statistics and Actuarial Science, The University of Iowa, USA, for assistance with R statistical programming. The authors also thank the anonymous reviewers for their constructive comments.

Funding Information

The financial support (grant reference No. MRP/3011/09) was from the University Grants Commission, New Delhi.


  1. Abbasi Z, Hooshyar S, Bagherieh-Najjar MB (2016) Improvement of callus production and shoot regeneration using various organs of soybean (Glycine max L. Merr) by response surface methodology. In Vitro Cell Dev Biol-Plant 52:537–545CrossRefGoogle Scholar
  2. Aileni M, Rao Kokkirala V, Reddy Kota S, Umate P, Abbagani S (2008) Efficient in-vitro regeneration from mature leaf explants of Scoparia dulcis L., an ethnomedicinal plant. J Herbs Spices Med Plants 14:200–207CrossRefGoogle Scholar
  3. Bansal M, Sudhakara Reddy M, Kumar A (2017) Optimization of cell growth and bacoside-a production in suspension cultures of Bacopa monnieri (L.) Wettst. using response surface methodology. In Vitro Cell Dev Biol-Plant 53:527–537CrossRefGoogle Scholar
  4. Burkill HM (2000) The useful plants of west tropical Africa, 2nd Ed, 5, Royal Botanic Gardens, Kew, pp 648Google Scholar
  5. Chakraborty D, Bandyopadhyay A, Bandopadhyay S, Gupta K, Chatterjee A (2010) Use of response surface methodology for optimization of a shoot regeneration protocol in Basilicum polystachyon. In Vitro Cell Dev Biol-Plant 46:451–459CrossRefGoogle Scholar
  6. Chong MN, Zhuc HY, Jin B (2010) Response surface optimization of photocatalytic process for degradation of Congo Red using H-titanate nanofiber catalyst. Chem Eng J 156:278–285CrossRefGoogle Scholar
  7. Davioud E, Kan C, Hamon J, Tempé J, Husson H-P (1989) Production of indole alkaloids by in vitro root cultures from Catharanthus trichophyllus. Phytochemistry 28:2675–2680CrossRefGoogle Scholar
  8. Elibol M (2004) Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology. Process Biochem 39:1057–1062CrossRefGoogle Scholar
  9. Hayashi T, Smith FT, Lee KH (1987) Antitumor agents. 89. Psychorubrin, a new cytotoxic naphthoquinone from Psychotria rubra and its structure-activity relationships. J Med Chem 30:2005–2008CrossRefGoogle Scholar
  10. Hayashi T, Okamura K, Kawasaki M, Morita N (1991) Two chemotypes of Scoparia dulcis in Paraguay. Phytochemistry 30:3617–3620CrossRefGoogle Scholar
  11. Hayashi T, Kawasaki M, Okamura K, Tamada Y, Morita N, Tezuka Y, Kikuchi T, Miwa Y, Taga T (1992) Scoparic acid A, a beta-glucuronidase inhibitor from Scoparia dulcis. J Nat Prod 55:1748–1755Google Scholar
  12. Hayashi T, Okamura K, Kawasaki M, Morita N (1993a) Production of diterpenoids by cultured cells from two chemotypes of Scoparia dulcis. Phytochemistry 33:353–356CrossRefGoogle Scholar
  13. Hayashi T, Okamura K, Tamada Y, Iida A, Fujita T, Morita N (1993b) A new chemotype of Scoparia dulcis. Phytochemistry 33:349–352CrossRefGoogle Scholar
  14. Hayashi T, Kasahara K, Sankawa U (1997) Efficient production of biologically active diterpenoids by leaf organ culture of Scoparia dulcis. Phytochemistry 46:517–520CrossRefGoogle Scholar
  15. Hayashi T, Asai T, Sankawa U (1999) Mevalonate-independent biosynthesis of bicyclic and tetracyclic diterpenes of Scoparia dulcis L. Tetrahedron Lett 40:8239–8243CrossRefGoogle Scholar
  16. Hirata K, Yamanaka A, Kurano N, Miyamoto K, Miura Y (1987) Production of indole alkaloids in multiple shoot culture of Catharanthus roseus (L). G. Don. Agric Biol Chem 51:1311–1317Google Scholar
  17. Krishnan PN, Decruse SW, Radha RK (2011) Conservation of medicinal plants of Western Ghats, India and its sustainable utilization through in vitro technology. In Vitro Cell Dev Biol-Plant 47:110–122CrossRefGoogle Scholar
  18. Kuhnt S, Rudak N (2013) Simultaneous optimization of multiple responses with the R package JOP. J Stat Softw 54:1–23CrossRefGoogle Scholar
  19. Latha M, Pari L, Sitasawad S, Bhonde R (2004) Insulin-secretagogue activity and cytoprotective role of the traditional antidiabetic plant Scoparia dulcis (Sweet Broomweed). Life Sci 75:2003–2014CrossRefGoogle Scholar
  20. Lee CL, Wang WL (1997) Biological statistics. Science Press, Beijing, ChinaGoogle Scholar
  21. Lenth RV (2009) Response-surface methods in R, using rsm. J Stat Softw 32:1–17CrossRefGoogle Scholar
  22. Liu Q, Yang Q-M, Hu H-J, Yang L, Yang Y-B, Chou G-X, Wang Z-T (2014) Bioactive diterpenoids and flavonoids from the aerial parts of Scoparia dulcis. J Nat Prod 77:1594–1600CrossRefGoogle Scholar
  23. Majumder S, Rahman MM, Bhadra SK (2011) Micropropagation of Scoparia dulcis Linn. through induction of indirect organogenesis. Asia Pac J Mol Biol Biotechnol 1:11–17Google Scholar
  24. Mukherjee PK (2003) Exploring botanicals in Indian system of medicine-regulatory perspectives. Clin Res Regul Aff 20:249–264CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Naveenchandra PM, Bhattacharya S, Ravishankar GA (2011) Culture media optimization through response surface methodology for in vitro shoot bud development of Solanum melongena L. for micropropagation. Int J Bioautomation 15:159–172Google Scholar
  27. Nkembo MK, Kurosaki F, Lee J-B, Hayashi T (2005a) Stimulation of calcium signal transduction results in enhancement of production of scopadulcic acid B by methyl jasmonate in the cultured tissues of Scoparia dulcis. Plant Biotechnol 22:333–337CrossRefGoogle Scholar
  28. Nkembo MK, Lee J-B, Hayashi T (2005b) Selective enhancement of scopadulcic acid B production in the cultured tissues of Scoparia dulcis by methyl jasmonate. Chem Pharm Bull 53:780–782CrossRefGoogle Scholar
  29. Osei-Safo D, Chama MA, Addae-Mensah I, Waibel R (2009) Hispidulin and other constituents of Scoparia dulcis Linn. J Sci Technol 29:7–15Google Scholar
  30. Pamunuwa G, Karunaratne N, Waisundara VY (2016) Antidiabetic properties, bioactive constituents, and other therapeutic effects of Scoparia dulcis. Evid Based Complement Alternat Med 8243215:1–11CrossRefGoogle Scholar
  31. Pari L, Venkateswaran S (2002) Hypoglycaemic activity of Scoparia dulcis L. extract in alloxan induced hyperglycaemic rats. Phytother Res 16:662–664CrossRefGoogle Scholar
  32. Premkumar G, Sankaranarayanan R, Jeeva S, Rajarathinam K (2011) Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L. (Scrophulariaceae)-an ethnomedicinal herb. Asian Pac J Trop Biomed 1:169–172CrossRefGoogle Scholar
  33. R Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL
  34. Rana B, Awasthi P, Kumbhar BK (2012) Optimization of processing conditions for cyanide content reduction in fresh bamboo shoot during NaCl treatment by response surface methodology. J Food Sci Technol 49:103–109CrossRefGoogle Scholar
  35. Rao Y, Lu S, Liu B, Tzeng Y (2006) Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation processes. Biochem Eng J 28:57–66CrossRefGoogle Scholar
  36. Srivastava S, Srivastava AK (2012) Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica. In Vitro Cell Dev Biol-Plant 48:73–84CrossRefGoogle Scholar
  37. Tabata M, Yamamoto H, Hiraoka N, Konoshima M (1972) Organization and alkaloid production in tissue cultures of Scopolia parviflora. Phytochemistry 11:949–955CrossRefGoogle Scholar
  38. Taylor L (2006) Vassourinha monograph 4/Scoparia dulcis. In: The rainforest, pharmacy to the world. Raintree Nutrition, Inc., Carson CityGoogle Scholar
  39. Wang Y, Fang X, An F, Wang G, Zhang X (2011) Improvement of antibiotic activity of Xenorhabdus bovienii by medium optimization using response surface methodology. Microb Cell Fact 10:98CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2020

Authors and Affiliations

  1. 1.Department of BotanyV.H.N. Senthikumara Nadar College (Autonomous)VirudhunagarIndia
  2. 2.Department of Horticultural Science, Faculty of AgriSciencesStellenbosch UniversityStellenboschSouth Africa
  3. 3.Department of BiotechnologyPondicherry UniversityPondicherryIndia
  4. 4.Department of MathematicsV.H.N. Senthikumara Nadar College (Autonomous)VirudhunagarIndia

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