Biomass and camptothecin production in the calcium chloride elicited and liquid medium overlayed Nothapodytes nimmoniana (J. Graham) Mabberly callus cultures

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Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
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Doi: 10.1007/s42535-021-00293-z
First Page: 104
Last Page: 114
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Keywords: Plant secondary metabolites, Phytochemicals, Elicitation, Calcium chloride, Cell culture, Liquid overlays


Nothapodytes nimmoniana (Icacinaceae) is an endangered medicinal plant species and a convenient source for the large-scale isolation of anti-cancer alkaloid camptothecin (CPT). Application of in vitro cultures elicitation is a promising strategy for enhancing the production of the alkaloid to obtain higher yield. In the present experiment, effects of calcium chloride elicitor on biomass and CPT production was studied at 10, 15, 20, 25, and 30 mM concentrations after 15, 30, and 45 days cultivation using solid and with a liquid medium overlay of Murashige and Skoog medium supplemented with N6-benzylaminopurine and naphthalene acetic acid (2.0 + 1.0 mg L−1). Maximum production of biomass and CPT was achieved with solid medium cultivation of the callus cultures overlayed with 1 mL liquid medium and CaCl2 elicitor at 25 mM. In these cultures, a maximum of 14.7-fold enhanced production of the alkaloid was achieved relative to the control. Higher concentrations of the CaCl2 elicitor resulted in a suppressive effect on the parameters studied relative to other treatment concentrations tested. The experimental results obtained suggest elicitation is an alternative strategy that could enhance production of CPT in the in vitro cultures of N. nimmoniana.

Plant secondary metabolites, Phytochemicals, Elicitation, Calcium chloride, Cell culture, Liquid overlays

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Aitken-Christie J, Jones C (1987) Towards automation: radiata pine shoot hedges in vitro. Plant Cell Tissue Organ Cult 8(3):185–196.

Ajungla L, Patil PP, Barmukh RB, Nikam TD (2009) Influence of biotic and abiotic elicitors on accumulation of hyoscyamine and scopolamine in the root cultures of Datura metel L. Indian J Biotechnol 8:317–322

Akula R, Ravishankar GA (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav 6(11):1720–1731.

Almagro L, Fernández-Pérez F, Pedreño MA (2015) Indole alkaloids from Catharanthus roseus: bioproduction and their effect on human health. Molecules 20(2):2973–3000.

Amin SA, Adhikari N, Jha T, Gayen S (2018) A review on camptothecin analogs with promising cytotoxic profile. Anti-Cancer Agents Med Chem 18(13):1796–1814.

Bagur R, Hajnóczky G (2017) Intracellular Ca2+ sensing: its role in calcium homeostasis and signaling. Mol Cell 66(6):780–788.

Baldi A, Singh D, Dixit VK (2008) Dual elicitation for the improved production of Withaferin A by the cell suspension cultures of Withania somnifera. Appl Biochem Biotechnol 151:556–564.

Berthouly M, Etienne H (2005) Temporary immersion system: new concept for use in liquid medium mass propagation. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for the in vitro plant propagation. Springer, Dordrecht, pp 165–195.

Bonfill M, Exposito O, Moyano E, Cusido RM, Palazo J, Pinol MT (2006) Manipulation by culture mixing and elicitation of paclitaxel and baccatin II production in Taxus baccata suspension cultures. In Vitro Cell Dev Biol Plant 42:422–426.

Bonfill M, Mangas S, Moyano E, Cusido RM, Palazon J (2011) Production of centellosides and phytosterols in cell suspension cultures of Centella asiatica. Plant Cell Tissue Organ Cult 104(1):61–67.

Bose J, Pottosin I, Shabala SSS, Palmgren MG, Shabala S (2011) Calcium efflux systems in stress signaling and adaptation in plants. Front Plant Sci 2:85.

Bramble JL, Graves DJ (1991) Calcium and phosphate effects on growth and alkaloid production in Coffea arabica: experimental results and mathematical model. Biotechnol Bioeng 37:859–868.

Carafoli E, Krebs J (2016) Why calcium? How calcium became the best communicator. J Biol Chem 291(40):20849–20857.

Cardillo AB, Talou JR, Giulietti AM (2016) Establishment, culture, and scale-up of Brugmansia candida hairy roots for the production of tropane alkaloids. Methods Mol Biol 1391:173–186.

Chandra S, Low PS (1997) Measurement of Ca2+ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells. J Biol Chem 272(45):28274–28280.

Dandin VS, Murthy HN (2012) Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semi-solid and liquid cultures and estimation of camptothecin in the regenerated plants. Acta Physiol Plant 34:1381–1386.

Deepthi S, Satheeshkumar K (2016) Enhanced camptothecin production induced by elicitors in the cell suspension cultures of Ophiorrhiza mungos Linn. Plant Cell Tissue Organ Cult 26(7):873–887.

Dicosmo F, Tallevi SG (1985) Plant cell cultures and microbial insult: interactions with biotechnological potential. Trends Biotechnol 3:110–111

Dmitriev A, Djatsok J, Grodzinsky D (1996) The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion. Plant Cell Rep 15(12):945–948.

Dutt M, Grosser JW (2010) An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus. Plant Cell Rep 29:1251–1260.

Fulzele DP, Satdive RK (2005) Comparison of techniques for the extraction of anti-cancer drug camptothecin from the Nothapodytes foetida. J Chromatogr Anal 1063(2):9–13.

Gontier E, Sangwan BS, Barbotin JN (1994) Effects of calcium, alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill. Plant Cell Rep 13:533–536.

Gopalakrishnan K, Krishnan S, Narayanan KP (2018) Tissue culture studies and estimation of camptothecin from Ophiorrhiza prostrata D. Don. Indian J Plant Physiol 23(3):582–592.

Gunter EA, Ovodov YS (2005) Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by the Silene vulgaris cell culture. J Biotechnol 117(4):385–393.

Harfi B, Khelifi-Slaoui M, Bekhouche M, Benyammi R, Hefferon K, Makhzoum A, Khelifi L (2016) Hyoscyamine production in hairy roots of three Datura species exposed to high-salt medium. In Vitro Cell Dev Biol Plant 52(1):92–98.

Hepler PK (2005) Calcium: a central regulator of plant growth and development. Plant Cell 17:2142–2155.

Hussein GMH, Abdel-Rahman TMA, Alwan AH (2016) Defense response enhancement in strawberry via elicitors. 3 Biotech 6:130.

Iranbakhsh AR, Oshagi MA, Ebadi M (2007) Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture. Pak J Biol Sci 10:1236–1242

Isah T (2015) Adjustments to in vitro culture conditions and associated anomalies in plants. Acta Biol Cracov Ser Bot 57(2):9–28.

Isah T (2017) Production of camptothecin in the elicited callus cultures of Nothapodytes nimmoniana (J. Graham) Mabberly. Chem Pap 71:1091–1106.

Isah T, Mujib A (2015a) In vitro propagation and camptothecin production in Nothapodytes nimmoniana. Plant Cell Tissue Organ Cult 121:1–10.

Isah T, Mujib A (2015b) Camptothecin from Nothapodytes nimmoniana: review on biotechnology applications. Acta Physiol Plant 37:106.

Isah T, Mujib A (2015c) Enhanced in vitro seedling recovery in Nothapodytes nimmoniana. Br Biotechnol J 6(1):2231–2927.

Isah T, Umar S, Mujib A et al (2018) Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. Plant Cell Tissue Organ Cult 132:239–265.

Jisha KG (2006) A study on production of camptothecin from the Ophiorrhiza mungos and Nothapodytes foetida using cell and tissue culture. Thesis submitted to Mahatma Gandhi University Through Amala Cancer Res Centre, Thrissur, in partial fulfillment of the requirements for the award of Doctor of Philosophy

Karwasara VS, Dixit VK (2013) Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabb. Plant Biotechnol Rep 12:1–13.

Klimaszewska K, Trontin JF, Becwar MR, Devillard C, Park YS, Lelu-Walter MA (2007) Recent progress in somatic embryogenesis of four Pinus spp. Tree for Sci Biotechnol 1(1):11–25 ((al-02669153))

Klüsener B, Young JJ, Murata Y, Allen GJ, Mori IC, Hugouvieux V, Schroeder JI (2002) Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells. Plant Physiol 130:2152–2163.

Knight MR, Cambel AK, Smith SM, Trewavas AJ (1991) Transgenic plant aequorin reports the effects of touch and cold shock and elicitors on cytoplasmic calcium. Nature 352:524–526.

Krishnakumar G, Dintu KP, Varghese SC, Nair DS, Gopinath G, Rameshkumar KB, Satheeshkumar K, Krishnan PN (2020) Ophiorrhiza, a promising herbaceous source of the anticancer compound camptothecin. Plant Sci Today 7(2):240–250.

Kudaka DM, Lodej PK, Baier A, Szyszka R (2016) Metalloproteinases of the extracellular matrix and their inhibitors. J Biotechnol Comput Biol Bionanotechnol 97(2):129–136.

Kulkarni AV, Patwardhan AA, Lele U, Malpathak NP (2010) Production of camptothecin in cultures of Chonemorpha grandiflora. Pharmacogn Res 2(5):296–299.

Lecourieux D, Mazars C, Pauly N, Ranjeva R, Pugin A (2002) Analysis and effects of cytosolic free calcium elevations in response to the elicitors in Nicotiana plumbaginifolia cells. Plant Cell 14:2627–2641.

Lecourieux D, Lamotte O, Bourque S, Wendehenne D, Mazars C, Ranjeva R, Pugin A (2005) Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells. Cell Calcium 38:527–538.

Lee-Parsons CWT, Ertürk S (2005) Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level. Plant Cell Rep 24:677–682.

Liu Z, Li Y, Yang T, Su J, Zhang M, Tian R, Li X, Pang Y, Qi J, Yang Y (2011) Shikonin accumulation is related to calcium homeostasis in Onosma paniculata cell cultures. Phyton Ann Rei Bot 51(1):103–113

Mahady GB, Liu C, Beecher WW (1998) Involvement of protein kinase and G proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis. Phytochemistry 48:93–102.

Mathur J (1991) Enhanced somatic embryogenesis in Selinum candolii DC. under a mineral oil overlay. Plant Cell Tissue Organ Cult 27:23–26

Mohamed GM, Amer AM, Osman NH, Sedikc MZ, Hussein MH (2021) Effects of different gelling agents on the different stages of rice regeneration in two rice cultivars. Saudi J Biol Sci.

Mujib A, Tonk D, Gulzar B, Maqsood M, Ali M (2020) Quantification of taxol by high-performance thin layer chromatography in Taxus wallichiana callus cultivated in vitro. J Biotechnol Comput Biol Bionanotechnol 102(4):337–347.

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497.

Naik PM, Al-Khayri JM (2016) Abiotic and biotic elicitors-role in secondary metabolites production through in vitro culture of medicinal plants. In: Shanker AK, Shanker C (eds) Abiotic and biotic stress in plants - Recent, advances and future perspectives. IntechOpen.

Ning W, Wang JX, Liu YM, Li N, Cao RQ (1998) The effects of Ca2+ during the elicitation of shikonin derivatives in Onosma paniculatum cells. In Vitro Cell Dev Biol Plant 34:261–265.

Osuna L, Moyano E, Mangas S, Bonfill M, Cusidó RM, Piñol MT, Zamilpa A, Tortoriello J, Palazón J (2008) Immobilization of Galphimia glauca plant cell suspensions for the production of enhanced amounts of Galphimine-B. Planta Med 74(01):94–99.

Osuna L, Tapia N, Zamilpa A, Jimenez-Ferrer E, Tortoriello J (2014) Biosynthesis stimulation of thenor-secotriterpene anxiolytics in the cell suspension cultures of Galphimia glauca. Cav Eng Life Sci 14(1):68–75.

Patwardhan A (2006) Domestication of Nothapodytes nimmoniana (grah.) Mabb. an endangered medicinal tree from Western Ghats of India. The Rufford Small Grants Foundation, UK, pp 7–15

Pitta-Alvarez SI, Spollansky TC, Giulietti AM (2000) The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in the hairy root cultures of Brugmansia candida. Enzyme Microb Technol 26:252–258.

Pommier Y (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789–802.

Prakash L, Middha SK, Mohanty SK, Swamy MK (2016) Micropropagation and validation of genetic and biochemicalfidelity among regenerants of Nothapodytes nimmoniana (Graham) Mabb. employing ISSR markers and HPLC. 3 Biotech 6:171.

Pullaiah T, Raghavendra PB, Karuppusamy S, Raveendran V, Anuradha M (eds) (2020) Camptothecin and camptothecin producing plants: botany, chemistry, anticancer activity and biotechnology. Academic Press/Elsevier Inc, UK/USA, pp 1–338.

Pullman GS, Skryabina A (2007) Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers. Plant Cell Rep 26(7):873–887.

Radman R, Saez T, Bucke C, Keshavarz T (2003) Elicitation of plants and microbial cell systems. Biotechnol Appl Biochem 37:91–102.

Roisin C, Gillet-Manceau F, Nava Saucedo JE, Fliniaux M, Jacquin-Dubreuil A, Barbotin JN (1997) Enhanced production of scopolin by the Solanum aviculare cells immobilized within the Ca-alginate gel beads. Plant Cell Rep 16(5):349–353.

Saniewski M, Horbowicz M, Saniewska A (2006) The effect of ruthenium red, a Ca2+ channel blocker, on a red pigment formation in mechanically wounded scales of Hippeastrum x hybr. hort., and on the growth and development of Phoma narcissi. J Fruit Ornamental Plant Res 14:211–222

Sano T, Higakib T, Handa K, Kadota Y, Kuchitsu K, Hasezawa S, Hoffmann A, Endter J, Zimmermann U, Hedrich R, Roitsch T (2006) Calcium ions are involved in delay of plant cell cycle progression by abiotic stresses. FEBS Lett 580:597–602.

Senapati SK (2015) A double phase culture system: an economic and time saving protocol for in vitro propagation of plant. SAJ Biotechnol 2:101.

Seybold H, Trempel F, Ranf S, Scheel D, Romeis T, Lee J (2014) Ca2+ signalling in plant immune response: from pattern recognition receptors to Ca2+ decoding mechanisms. New Phytol 204(4):782–790.

Sharma A, Angulo-Bejarano PI, Madariaga-Navarrete A, Oza G, Iqbal H, Cardoso-Taketa A, Luisa Villarreal M (2018) Multidisciplinary investigations on Galphimia glauca: a Mexican medicinal plant with pharmacological potential. Molecules 23(11):2985.

Siddiqui ZH, Mujib A (2011) Accumulation of vincristine in calcium chloride elicitated Catharanthus roseus cultures. Nat Prod J 2(4):307–315.

Siddiqui ZH, Mujib A, Maqsood M (2011) Liquid overlaying improves somatic embryogenesis in Catharanthus roseus. Plant Cell Tissue Organ Cult 104:247–256.

Sirikantaramas S, Yamazaki M, Saito K (2013) Camptothecin: biosynthesis, biotechnological production and resistance mechanism(s). Adv Bot Res 68:139–161.

Song WY, Zhang ZB, Shao HB, Guo XL, Cao HX, Zhao HB, Fu ZY, Hu XJ (2008) Relationship between calcium decoding elements and plant abiotic-stress resistance. Int J Biol Sci 4:116–125.

Sudha G, Ravishankar GA (2003) Elicitation of anthocyanin production in callus cultures of Daucus carota and involvement of calcium channel modulators. Curr Sci 84(6):775–779

Suhas S, Ramesha BT, Ravikanth G, Gunaga RP, Vasudeva R, Ganeshaiah KN, Shaanker RU (2007) Chemical profiling of Nothapodytes nimmoniana populations in the Western Ghats, India for anticancer compound Camptothecin. Curr Sci 92:1142–1147

Trewavas AJ (2000) Signal perception and transduction. In: Buchanan B, Gruissem W, Jones R (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologist, Rockville, pp 930–987

Tuteja N, Mahajan S (2007) Calcium signaling network in plants: an overview. Plant Signal Behav 2(2):79–85.

White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92(4):487–511.

Zafar N, Mujib A, Ali M, Tonk D, Gulzar B, Malik MQ, Mamgain J, Sayeed R (2020) Cadmium chloride (CdCl2) elicitation improves reserpine and ajmalicine yield in Rauvolfia serpentina as revealed by high-performance thin-layer chromatography (HPTLC). 3 Biotech 10:344.

Zhang L, Du L, Poovaiah BW (2014) Calcium signaling and biotic defense responses in plants. Plant Signal Behav 9(11):e973818.

Zhao J, Sakai K (2003) Multiple signalling pathways mediate fungal elicitor-induced β-thujaplicin biosynthesis in Cupressus lusitanica cell cultures. J Exp Bot 54(383):647–656.

Zhao J, Fujita K, Sakai K (2001a) Production of β-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes. Biosci Biotechnol Biochem 65(5):1027–1032.

Zhao J, Hu Q, Guo YQ, Zhu WH (2001b) Elicitor-induced indole alkaloid biosynthesis in Catharanthus roseus cell cultures is related to Ca2+ influx and the oxidative burst. Plant Sci 161:423–431.

Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23(4):283–333.



Authors are grateful to Jamia Hamdard New Delhi, India for support to the research work.

Author Information

Isah Tasiu
Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India
Masood Samiya
Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India

Umar Shahid
Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India