In Vitro Plant Cell Cultures: A Route to Production of Natural Molecules and Systematic In Vitro Assays for their Biological Properties

  • Peeyushi Verma
  • Rakhi ChaturvediEmail author


Nature provides an array of medicinal plants that serve the society by treating a wide range of diseases. Many plants are on the verge of extinction due to their extensive use, ignorance, and restricted habitat. Thus, generation of in vitro cultures via “plant tissue culture techniques” is necessary for maintenance and large-scale propagation of flora. Additionally, these in vitro cultures also provide constant production of a wide variety of complex and structurally diverse secondary metabolites, possessing a range of therapeutic properties and biological activities. This chapter emphasizes on the establishment of in vitro plant cell cultures for secondary metabolite production and their applications in various biological assays. Furthermore, a brief description of in vitro assays is given to elucidate a particular type of bioactivity and its mechanism of action.


Bioactivity In vitro Plant cell cultures Secondary metabolites 


  1. Anulika NP, Ignatius EO, Raymond ES, Osasere O-I, Abiola HA (2016) The chemistry of natural product: plant secondary metabolites. Int J Technol Enhanc Emerg Eng Res 4:1–8Google Scholar
  2. Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating antimicrobial activity: a review. J Pharmaceut Anal 6:71–79Google Scholar
  3. Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 239:70–76PubMedCrossRefGoogle Scholar
  4. Bidlack WR (2000) Phytochemicals as bioactive agents. Technomic Publishers, Lancaster, PACrossRefGoogle Scholar
  5. Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181:1199CrossRefGoogle Scholar
  6. Caruso JL, Callahan J, DeChant C, Jayasimhulu K, Winget GD (2000) Carnosic acid in green callus and regenerated shoots of Rosmarinus officinalis. Plant Cell Rep 19:500–503PubMedCrossRefGoogle Scholar
  7. Chattopadhyay S, Srivastava AK, Bisaria VS (2002) Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum. Appl Biochem Biotechnol 102–103:381–393PubMedCrossRefGoogle Scholar
  8. Chen S-A, Wang X, Zhao B, Yuan X, Wang Y (2003) Production of crocin using Crocus sativus callus by two-stage culture system. Biotechnol Lett 25:1235–1238PubMedCrossRefGoogle Scholar
  9. Choma IM, Grzelak EM (2011) Bioautography detection in thin-layer chromatography. J Chromatogr A 1218:2684–2691PubMedCrossRefGoogle Scholar
  10. Edahiro J, Yamada M, Seike S, Kakigi Y, Miyanaga K, Nakamura M, Kanamori T, Seki M (2005) Separation of cultured strawberry cells producing anthocyanins in aqueous two-phase system. J Biosci Bioeng 100:449–454PubMedCrossRefGoogle Scholar
  11. Falahi H, Sharifi M, Maivan HZ, Chashmi NA (2017) Phenylethanoid glycosides accumulation in roots of Scrophularia striata as a response to water stress. Environ Exp Bot 147:13–21CrossRefGoogle Scholar
  12. Garrat DC (1964) The quantitative analysis of drugs, Japan. Chapman and Hall, JapanGoogle Scholar
  13. Georgiev MI, Kuzeva SL, Pavlov AI, Kovacheva EG, Ilieva MP (2007) Elicitation of rosmarinic acid by Lavandula vera MM cell suspension culture with abiotic elicitors. World J Microbiol Biotechnol 23:301–304CrossRefGoogle Scholar
  14. Gomes de Melo J, de Sousa Araujo TA, Nobre T, de Almeida e Castro V, Lyra de Vasconcelos Cabral D, do Desterro Rodrigues M, Carneiro do Nascimento S, Cavalcanti de Amorim EL, de Albuquerque UP (2010) Antiproliferative activity, antioxidant capacity and tannin content in plants of semi-arid northeastern Brazil. Molecules 15:8534–8542PubMedPubMedCentralCrossRefGoogle Scholar
  15. Gray AM, Flatt PR (1997) Nature’s own pharmacy: the diabetes perspective. Proc Nutr Soc 56:507–517PubMedCrossRefGoogle Scholar
  16. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126:131–138PubMedCrossRefGoogle Scholar
  17. Griffits JB (1985) Advances in biotechnological processes: volume 2. FEBS Lett 188:168–169CrossRefGoogle Scholar
  18. Grzegorczyk I, Krolicka A, Wysokinska H (2006) Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid. Z Naturforsch C 61:351–356PubMedCrossRefGoogle Scholar
  19. Grzegorczyk I, Matkowski A, Wysokińska H (2007) Antioxidant activity of extracts from in vitro cultures of Salvia officinalis L. Food Chem 104:536–541CrossRefGoogle Scholar
  20. Gülçın İ, Oktay M, Kıreçcı E, Küfrevıoǧlu Öİ (2003) Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem 83:371–382CrossRefGoogle Scholar
  21. Guschin A, Ryzhikh P, Rumyantseva T, Gomberg M, Unemo M (2015) Treatment efficacy, treatment failures and selection of macrolide resistance in patients with high load of Mycoplasma genitalium during treatment of male urethritis with josamycin. BMC Infect Dis 15:40PubMedPubMedCentralCrossRefGoogle Scholar
  22. Halliwell B, Gutteridge JM (1995) The definition and measurement of antioxidants in biological systems. Free Radic Biol Med 18:125–126PubMedCrossRefGoogle Scholar
  23. Halliwell B, Gutteridge JM, Aruoma OI (1987) The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165:215–219PubMedCrossRefGoogle Scholar
  24. Hartmann T (1999) Chemical ecology of pyrrolizidine alkaloids. Planta 207:483–495CrossRefGoogle Scholar
  25. Hussain MS, Fareed S, Ansari S, Rahman MA, Ahmad IZ, Saeed M (2012) Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 4:10–20PubMedPubMedCentralCrossRefGoogle Scholar
  26. Jayaprakasha GK, Jaganmohan Rao L, Sakariah KK (2004) Antioxidant activities of flavidin in different in vitro model systems. Bioorg Med Chem 12:5141–5146PubMedCrossRefGoogle Scholar
  27. Johnson M, Wesely EG, Zahir Hussain MI, Selvan N (2010) In vivo and in vitro phytochemical and antibacterial efficacy of Baliospermum montanum (Wïlld.) Muell. Arg. Asian Pac J Trop Med 3:894–897CrossRefGoogle Scholar
  28. Kaminaga Y, Nagatsu A, Akiyama T, Sugimoto N, Yamazaki T, Maitani T, Mizukami H (2003) Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus. FEBS Lett 555:311–316PubMedCrossRefGoogle Scholar
  29. Karban R, Baldwin IT (1997) Induced plant responses to herbivory. University of Chicago Press, Chicago, p 1997Google Scholar
  30. Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291:2141–2144PubMedCrossRefPubMedCentralGoogle Scholar
  31. Kim HK, Verpoorte R (2010) Sample preparation for plant metabolomics. Phytochem Anal 21:4–13PubMedCrossRefGoogle Scholar
  32. Kuete V, Wabo HK, Eyong KO, Feussi MT, Wiench B, Krusche B, Tane P, Folefoc GN, Efferth T (2011) Anticancer activities of six selected natural compounds of some cameroonian medicinal plants. PLoS One 6:e21762PubMedPubMedCentralCrossRefGoogle Scholar
  33. Kunchandy E, Rao MNA (1990) Oxygen radical scavenging activity of curcumin. Int J Pharm 58:237–240CrossRefGoogle Scholar
  34. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420PubMedCrossRefGoogle Scholar
  35. Lazar T (2003) Taiz, L. and Zeiger, E. Plant physiology. 3rd edn. Ann Bot 91:750–751PubMedCentralCrossRefGoogle Scholar
  36. Matkowski A (2008) Plant in vitro culture for the production of antioxidants—a review. Biotechnol Adv 26:548–560PubMedCrossRefGoogle Scholar
  37. Mayers DL, Sobel JD, Ouellette M, Kaye KS, Marchaim D (2009) Antimicrobial drug resistance: clinical and epidemiological aspects. Springer, Dordrecht, Heidelberg, LondonCrossRefGoogle Scholar
  38. McCauley J, Zivanovic A, Skropeta D (2013) Bioassays for anticancer activities. Methods Mol Biol 1055:191–205PubMedCrossRefPubMedCentralGoogle Scholar
  39. Mensor LL, Menezes FS, Leitao GG, Reis AS, dos Santos TC, Coube CS, Leitao SG (2001) Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 15:127–130PubMedCrossRefGoogle Scholar
  40. Meyer AS, Isaksen A (1995) Application of enzymes as food antioxidants. Trends Food Sci Technol 6:300–304CrossRefGoogle Scholar
  41. Miura H, Kitamura Y, Ikenaga T, Mizobe K, Shimizu T, Nakamura M, Kato Y, Yamada T, Maitani T, Goda Y (1998) Anthocyanin production of Glehnia littoralis callus cultures. Phytochemistry 48:279–283PubMedCrossRefGoogle Scholar
  42. Mulabagal Vanisree M, Lee C-Y, Lo S-F, Nalawade SM, Lin CY, Tsay H-S (2004) Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Botan Bull Acad Sin 45:1–22Google Scholar
  43. Nishikimi M, Appaji Rao N, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46:849–854PubMedCrossRefGoogle Scholar
  44. Noro T, Oda Y, Miyase T, Ueno A, Fukushima S (1983) Inhibitors of xanthine oxidase from the flowers and buds of Daphne genkwa. Chem Pharm Bull 31:3984–3987PubMedCrossRefGoogle Scholar
  45. O’Connell JE, Fox PF (2001) Significance and applications of phenolic compounds in the production and quality of milk and dairy products: a review. Int Dairy J 11:103–120CrossRefGoogle Scholar
  46. Parveen A, Chakraborty A, Konreddy AK, Chakravarty H, Sharon A, Trivedi V, Bal C (2013) Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of alpha-pyrone analogs as anti-malarial agent. Eur J Med Chem 70:607–612PubMedCrossRefGoogle Scholar
  47. Pavlov A, Georgiev M, Bley T (2007) Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor. Z Naturforsch C 62:439–446PubMedCrossRefGoogle Scholar
  48. Petrovska B (2012a) Historical review of medicinal plants’ usage. Pharmacogn Rev 6:1–5PubMedPubMedCentralCrossRefGoogle Scholar
  49. Petrovska BB (2012b) Historical review of medicinal plants’ usage. Pharmacogn Rev 6:1–5PubMedPubMedCentralCrossRefGoogle Scholar
  50. Prakash G, Srivastava AK (2006) Modeling of azadirachtin production by Azadirachta indica and its use for feed forward optimization studies. Biochem Eng J 29:62–68CrossRefGoogle Scholar
  51. Prior RL, Wu X, Schaich K (2005) Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53:4290–4302PubMedCrossRefGoogle Scholar
  52. Qin L, Markham KR, Paré PW, Dixon RA, Mabry TJ (1993) Flavonoids from elicitor-treated cell suspension cultures of Cephalocereus senilis. Phytochemistry 32:925–928CrossRefGoogle Scholar
  53. Rajaram K, Moushmi M, Velayutham Dass Prakash M, Kumpati P, Ganasaraswathi M, Sureshkumar P (2013) Comparative bioactive studies between wild plant and callus culture of tephrosia tinctoria pers. Appl Biochem Biotechnol 171:2105–2120PubMedCrossRefGoogle Scholar
  54. Rajendran R, Narashimman BS, Trivedi V, Chaturvedi R (2017) Isolation and quantification of antimalarial N-alkylamides from flower-head derived in vitro callus cultures of Spilanthes paniculata. J Biosci Bioeng 124:99–107PubMedCrossRefGoogle Scholar
  55. Rich SM, Leendertz FH, Xu G, LeBreton M, Djoko CF, Aminake MN, Takang EE, Diffo JL, Pike BL, Rosenthal BM, Formenty P, Boesch C, Ayala FJ, Wolfe ND (2009) The origin of malignant malaria. Proc Natl Acad Sci U S A 106:14902–14907PubMedPubMedCentralCrossRefGoogle Scholar
  56. Robak J, Gryglewski RJ (1988) Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 37:837–841PubMedCrossRefGoogle Scholar
  57. Rosenthal GA, Berenbaum MR (1991) Herbivores, their interactions with secondary plant metabolites. Academic Press, San Diego, CAGoogle Scholar
  58. Ruch RJ, Cheng SJ, Klaunig JE (1989) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10:1003–1008PubMedCrossRefGoogle Scholar
  59. Salazar R, Pozos ME, Cordero P, Perez J, Salinas MC, Waksman N (2008) Determination of the antioxidant activity of plants from Northeast Mexico. Pharm Biol 46:166–170CrossRefGoogle Scholar
  60. Sampaio BL, Edrada-Ebel R, Da Costa FB (2016) Effect of the environment on the secondary metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants. Sci Rep 6:29265PubMedPubMedCentralCrossRefGoogle Scholar
  61. Sanchez-Sampedro MA, Fernandez-Tarrago J, Corchete P (2005) Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaertn. J Biotechnol 119:60–69PubMedCrossRefGoogle Scholar
  62. Schmeda-Hirschmann G, Razmilic I, Sauvain M, Moretti C, Muñoz V, Ruiz E, Balanza E, Fournet A (1996) Antiprotozoal activity of Jatrogrossidione from Jatropha grossidentata and Jatrophone from Jatropha isabellii. Phytother Res 10:375–378CrossRefGoogle Scholar
  63. Sharma RA, Steward WP, Gescher AJ (2007) Pharmacokinetics and pharmacodynamics of curcumin. Adv Exp Med Biol 595:453–470PubMedCrossRefGoogle Scholar
  64. Shimada K, Fujikawa K, Yahara K, Nakamura T (1992) Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem 40:945–948CrossRefGoogle Scholar
  65. Singh M, Roy B, Tandon V, Chaturvedi R (2014) Extracts of dedifferentiated cultures of Spilanthes acmella Murr. possess antioxidant and anthelmintic properties and hold promise as an alternative source of herbal medicine. Plant Biosyst 148:259–267CrossRefGoogle Scholar
  66. Sreejayan, Rao MN (1997) Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol 49:105–107PubMedCrossRefGoogle Scholar
  67. Srivastava P, Kasoju N, Bora U, Chaturvedi R (2009) Dedifferentiation of leaf explants and cytotoxic activity of an aqueous extract of cell cultures of Lantana camara L. Plant Cell Tissue Organ Cult 99:1–7CrossRefGoogle Scholar
  68. Srivastava P, Kasoju N, Bora U, Chaturvedi R (2010) Accumulation of betulinic, oleanolic, and ursolic acids in In vitro cell cultures of Lantana camara L. and their significant cytotoxic effects on HeLa cell lines. Biotechnol Bioprocess Eng 15:1038–1046CrossRefGoogle Scholar
  69. Thummel RP (1979) The basis of organic chemistry. Second Edition (Fessenden, Ralph J.; Fessenden, Joan S.). J Chem Educ 56:A144CrossRefGoogle Scholar
  70. Umesh TG (2014) In vitro callus induction and antioxidant potential of Decalepis hamiltonii (wight and arn). Int J Pharm Pharm Sci 6:452–456Google Scholar
  71. Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25CrossRefGoogle Scholar
  72. Vijayalakshmi K, Selvaraj CI, Sivalingam S, Arumugam P (2014) In vitro investigation of antidiabetic potential of selected traditional medicinal plants. Int J Pharmacogn Phytochem Res 6:856–861Google Scholar
  73. Wickramaratne MN, Punchihewa JC, Wickramaratne DBM (2016) In-vitro alpha amylase inhibitory activity of the leaf extracts of Adenanthera pavonina. BMC Complement Altern Med 16:466PubMedPubMedCentralCrossRefGoogle Scholar
  74. Wink M (2015) Modes of action of herbal medicines and plant secondary metabolites. Fortschr Med 2:251Google Scholar
  75. Zhang MH, Lu F, Cao J, Gao Q (2015) Comparative study of assay methods for in vitro antimalarial drug efficacy testing in Plasmodium falciparum. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi 27:146–151PubMedPubMedCentralGoogle Scholar
  76. Zhong JJ (2001) Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures. In: Zhong JJ, Byun SY, Cho GH, Choi JW, Haigh JR, Honda H, James E, Kijne JW, Kim DI, Kobayashi T, Lee JM, Kino-oka M, Linden JC, Liu C, Memelink J, Mirjalili N, Nagatome H, Taya M, Phisaphalong M, van der Heijden R, Verpoorte R (eds) Plant cells. Springer, Berlin, HeidelbergCrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Indian Institute of Technology GuwahatiGuwahatiIndia

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