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Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana

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Abstract

For the purpose of enhancing the secondary metabolite content in micropropagated Stevia rebaudiana plants without inhibiting plant growth, node explants were cultured on woody plant medium (WPM) containing alginate (ALG), casein hydrolysate (CH), pectin (PEC), yeast extract (YE), methyl jasmonate (MeJA), salicylic acid (SA), or chitosan (CHI). The highest shoot number; shoot length, node number, and leaf number; leaf length; and stem diameter were observed on WPM containing 1.0 g/L YE; 100 µM CHI; 0.5 g/L CH; and 1.0 g/L ALG, respectively. The root regeneration frequency reached 100 % on WPM supplemented with 0.5, 1.0, or 2.0 g/L PEC, 1.0 g/L YE, or 50 µM CHI and the control. The highest root number was obtained on WPM containing 0.5 g/L PEC, while the longest root length was observed on WPM containing 1.0 g/L YE. The highest biomass accumulation was observed with treatment of 100 µM CHI. According to the high-performance liquid chromatography results, except for the treatments with 200 µM CHI, 100 or 200 µM MeJA, and 200 µM SA, the remaining elicitor treatments increased stevioside production compared to the control. The production of stevioside increased from 1.56 mg/g dry weight (DW) to 14.69 and 14.54 mg/g DW in the in vitro plantlets exposed to 0.5 g/L ALG and 2.0 g/L YE, respectively. Rebaudioside A was observed on only 0.5 g/L ALG-treated plants as 0.55 mg/g DW. The stevioside content of field-grown plants was identified as 15.06 mg/g DW. The present findings provide important information regarding the effect of elicitors on plant growth and secondary metabolite production of in vitro micropropagated S. rebaudiana.

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References

  • Ait Barka E, Eullaffroy P, Clément C, Vernet G (2004) Chitosan improves development, and protects Vitis vinifera L. against Botrytis cinerea. J Plant Cell Rep 22(8):608–614

    Article  CAS  Google Scholar 

  • Akimoto C, Aoyagi H, Tanaka H (1999) Endogenous elicitor-like effects of alginate on physiological activities of plant cells. Appl Microbiol Biotechnol 52:429–436

    Article  CAS  Google Scholar 

  • Aman N, Hadi F, Khalil SA, Zamir R, Ahmad N (2013) Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni). C R Biol 336:486–492

    Article  CAS  PubMed  Google Scholar 

  • Angelova Z, Georgiev S, Roos W (2006) Elicitation of plants. Biotechnol Biotechnol Equip 20(2):72–83. doi:10.1080/13102818.2006.10817345

    Article  CAS  Google Scholar 

  • Baenas N, García-Viguera C, Moreno DA (2014) Elicitation: a tool for enriching the bioactive composition of foods. Molecules 19:13541–13563. doi:10.3390/molecules190913541

    Article  PubMed  Google Scholar 

  • Benhamou N, Thériault G (1992) Treatment with chitosan enhances resistance of tomato plants to the crown and root rot pathogen Fusarium oxysporum f. sp. radicis-lycopersici. Physiol Mol Plant Pathol 41(1):33–52. doi:10.1016/0885-5765(92)90047-Y

    Article  CAS  Google Scholar 

  • Bondarev N, Reshetnyak O, Nosov A (2001) Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni. Plant Sci 161:155–163. doi:10.1016/S0168-9452(01)00400-9

    Article  CAS  Google Scholar 

  • Brandle JE, Rosa N (1992) Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Can J Plant Sci 72:1263–1266

    Article  CAS  Google Scholar 

  • Brandle JE, Telmer PG (2007) Steviol glycoside biosynthesis. Phytochemistry 68:1855–1863. doi:10.1016/j.phytochem.2007.02.010

    Article  CAS  PubMed  Google Scholar 

  • Brandle JE, Starratt AN, Gijzen M (1998) Stevia rebaudiana: its agricultural, biological and chemical properties. Can J Plant Sci 78:527–536. doi:10.4141/P97-114

    Article  CAS  Google Scholar 

  • Brandle JE, Richman A, Swanson AK, Chapman BP (2002) Leaf ESTs from Stevia rebaudiana: a resource for gene discovery in diterpene synthesis. Plant Mol Biol 50:613–622. doi:10.1023/A:1019993221986

    Article  CAS  PubMed  Google Scholar 

  • Cai Z, Kastell A, Mewis I, Knorr D, Smetansk I (2012) Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of Vitis vinifera. Plant Cell Tissue Organ Cult 108:401–409. doi:10.1007/s11240-011-0051-3

    Article  CAS  Google Scholar 

  • Ceunen S, Geuns JMC (2013) Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Sci 198:72–82. doi:10.1016/j.plantsci.2012.10.003

    Article  CAS  PubMed  Google Scholar 

  • Dey A, Kundu S, Bandyopadhyay A, Bhattacharjee A (2013) Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni. C R Biol 336:17–28. doi:10.1016/j.crvi.2012.11.007

    Article  CAS  PubMed  Google Scholar 

  • Dörnenburg H, Knorr D (1995) Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzyme Microb Technol 17:674–684

    Article  Google Scholar 

  • Gadzovska Simic S, Tusevski O, Maury S, Delaunay A, Joseph C, Hagège D (2014) Effects of polysaccharide elicitors on secondary metabolite production and antioxidant response in Hypericum perforatum L. shoot cultures. Sci World J 2014:609649. doi:10.1155/2014/609649

    Article  Google Scholar 

  • Gardana C, Scaglianti M, Simonetti P (2010) Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry. J Chromatogr A 1217:1463–1470. doi:10.1016/j.chroma.2009.12.036

    Article  CAS  PubMed  Google Scholar 

  • Gupta P, Sharma S, Saxena S (2014) Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for steviol glycoside production. Appl Biochem Biotechnol 172(6):2894–2906. doi:10.1007/s12010-014-0736-2

    Article  CAS  PubMed  Google Scholar 

  • Gupta P, Sharma S, Saxena S (2015) Biomass yield and steviol glycoside production in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol. Appl Biochem Biotechnol 176(3):863–874. doi:10.1007/s12010-015-1616-0

    Article  CAS  PubMed  Google Scholar 

  • Gupta P, Sharma S, Saxena S (2016) Effect of abiotic stress on growth parameters and steviol glycoside content in Stevia rebaudiana (Bertoni) raised in vitro. J Appl Res Med Aromat Plants. doi:10.1016/j.jarmap.2016.03.004

    Google Scholar 

  • Hsing YI, Su WF, Chang WC (1983) Accumulation of stevioside and rebaudioside A in callus culture of Stevia rebaudiana Bertoni. Bot Bull Acad Sin 24:115–119

    CAS  Google Scholar 

  • Humphrey TV, Richman AS, Menassa R, Brandle JE (2006) Spatial organisation of four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Mol Biol 61:47–62. doi:10.1007/s11103-005-5966-9

    Article  CAS  PubMed  Google Scholar 

  • Khalil SA, Kamal N, Sajid M, Ahmad N, Zamir R, Ahmad N, Ali S (2016) Synergism of polyamines and plant growth regulators enhanced morphogenesis, stevioside content, and production of commercially important natural antioxidants in Stevia rebaudiana Bert. In Vitro Cell Dev Biol Plant 52:174–184. doi:10.1007/s11627-016-9749-6

    Article  CAS  Google Scholar 

  • Kim OT, Kim MY, Hong MH, Ahn JC, Hwang B (2004) Stimulation of asiaticoside accumulation in the whole plant cultures of Centella asiatica (L.) Urban by elicitors. Plant Cell Rep 23:339–344. doi:10.1007/s00299-004-0826-7

    Article  CAS  PubMed  Google Scholar 

  • Ladygin VG, Bondarev NI, Semenova GA, Smolov AA, Reshetnyak OV, Nosov AM (2008) Chloroplast ultrastructure, photosynthetic apparatus activities and production of steviol glycosides in Stevia rebaudiana in vivo and in vitro. Biol Plant 52:9–16. doi:10.1007/s10535-008-0002-y

    Article  CAS  Google Scholar 

  • Li L, Zhang CR (2006) Production of puerarin and isoflavones in cell suspension cultures of Pueraria lobata (Willd.): effects of medium supplementation with casein hydrolysate and coconut milk. J Environ Biol 27(1):21–26

    PubMed  Google Scholar 

  • Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Int Plant Prop Soc 30:421–427

    Google Scholar 

  • Mathur S, Shekhawat GS (2013) Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside. Acta Physiol Plant 35:931–939. doi:10.1007/s11738-012-1136-2

    Article  CAS  Google Scholar 

  • Muffler K, Leipold D, Scheller MC, Haas C, Steingroewer J, Bley T, Neuhaus HE, Mirata MA, Schrader J, Ulber R (2011) Biotransformation of triterpenes. Process Biochem 46:1–15

    Article  CAS  Google Scholar 

  • Murthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult 118:1–16. doi:10.1007/s11240-014-0467-7

    Article  CAS  Google Scholar 

  • Nanayakkara NP, Klocke JA, Compadre CM, Hussain RA, Pezzuto JM, Kinghorn AD (1987) Characterization and feeding deterrent effects on the aphid, Schizaphis graminum, of some derivatives of the sweet compounds, stevioside and rebaudioside A. J Nat Prod 50(3):434–441. doi:10.1021/np50051a015

    Article  CAS  PubMed  Google Scholar 

  • Nge KL, New N, Chandrkrachang S, Stevens WF (2006) Chitosan as a growth stimulator in orchid tissue culture. Plant Sci 170:1185–1190

    Article  CAS  Google Scholar 

  • Pandey H, Pandey P, Pandey SS, Singh S, Banerjee S (2016) Meeting the challenge of stevioside production in the hairy roots of Stevia rebaudiana by probing the underlying process. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-016-1020-7

    Google Scholar 

  • Pérez-Alonso N, Capote A, Gerth A, Jiménez E (2012) Increased cardenolides production by elicitation of Digitalis lanata shoots cultured in temporary immersion systems. Plant Cell Tissue Organ Cult 110:153–162. doi:10.1007/s11240-012-0139-4

    Article  Google Scholar 

  • Pornpienpakdee P, Singhasurasak R, Chaiyasap P, Pichyangkura R, Bunjongrat R, Chadchawan S, Limpanavech P (2010) Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan. Sci Hortic 124:490–499. doi:10.1016/j.scienta.2010.02.008

    Article  CAS  Google Scholar 

  • Putalun W, Udomsin O, Yusakul G, Juengwatanatrakul T, Sakamoto S, Tanaka H (2010) Enhanced plumbagin production from in vitro cultures of Drosera burmanii using elicitation. Biotechnol Lett 32:721–724. doi:10.1007/s10529-010-0202-3

    Article  CAS  PubMed  Google Scholar 

  • Ramakrishna A, Ravishankar GA (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav 6(11):1720–1731. doi:10.4161/psb.6.11.17613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reis RV, Borges APPL, Chierrito TPC, de Souto ER, de Souza LM, Iacomini M, de Oliveira AJB, Gonçalves RAC (2011) Establishment of adventitious root culture of Stevia rebaudiana Bertoni in a roller bottle system. Plant Cell Tissue Organ Cult 106:329–335. doi:10.1007/s11240-011-9925-7

    Article  Google Scholar 

  • Rhee HS, Cho HY, Son SY, Yoon SYH, Park JM (2010) Enhanced accumulation of decursin and decursinol angelate in root cultures and intact roots of Angelica gigas Nakai following elicitation. Plant Cell Tissue Organ Cult 101:295–302. doi:10.1007/s11240-010-9688-6

    Article  CAS  Google Scholar 

  • Richman AS, Gijzen M, Starratt AN, Yang Z, Brandle JE (1999) Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway. Plant J 19(4):411–421. doi:10.1046/j.1365-313X.1999.00531.x

    Article  CAS  PubMed  Google Scholar 

  • Shabani L, Ehsanpour AA, Asghari G, Emami J (2009) Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid. Russ J Plant Physiol 56(5):621–626. doi:10.1134/S1021443709050069

    Article  CAS  Google Scholar 

  • Sharma M, Sharma A, Kumar A, Kumar Basu S (2011) Enhancement of secondary metabolites in cultured plant cells through stress stimulus. Am J Plant Physiol 6(2):50–71. doi:10.3923/ajpp.2011.50.71

    Article  CAS  Google Scholar 

  • Sharma M, Ahuja A, Gupta R, Mallubhotla S (2015) Enhanced bacoside production in shoot cultures of Bacopa monnieri under the influence of abiotic elicitors. Nat Prod Res 29(8):745–749. doi:10.1080/14786419.2014.986657

    Article  CAS  PubMed  Google Scholar 

  • Singh SD, Rao GP (2005) Stevia: the herbal sugar of 21st century. Sugar Tech 7(l):17–24

    Article  CAS  Google Scholar 

  • Singh P, Dwivedi P, Atri N (2014) In vitro shoot multiplication of Stevia and assessment of stevioside content and genetic fidelity of the regenerants. Sugar Tech 16(4):430–439. doi:10.1007/s12355-013-0292-z

    Article  CAS  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114:121–129. doi:10.1007/s11240-013-0297-z

    Article  CAS  Google Scholar 

  • Sivaram L, Mukundan U (2003) In vitro culture studies on Stevia rebaudiana. In Vitro Cell Dev Biol Plant 39:520–523. doi:10.1079/IVP2003438

    Article  Google Scholar 

  • Skrzypczak-Pietraszek E, Słota J, Pietraszek J (2014) The influence of l-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. ex Regel shoot culture. Acta Biochim Pol 61(1):47–53

    PubMed  Google Scholar 

  • Sridhar TM, Aswath CR (2014) Influence of additives on enhanced in vitro shoot multiplication of Stevia rebaudiana (Bert.)—An important anti diabetic medicinal plant. Am J Plant Sci 5:192–199

    Article  Google Scholar 

  • Thaweesak J, Seiichi S, Hiroyuki T, Waraporn P (2011) Elicitation effect on production of plumbagin in in vitro culture of Drosera indica L. J Med Plants Res 5(19):4949–4953

    Google Scholar 

  • Tocci N, Ferrari F, Santamaria AR, Valletta A, Rovardi I, Pasqua G (2010) Chitosan enhances xanthone production in Hypericum perforatum subsp. angustifolium cell cultures. Nat Prod Res 24(3):286–293. doi:10.1080/14786410903006353

    Article  CAS  PubMed  Google Scholar 

  • Vasconsuelo A, Giuletti AM, Picotto G, Rodriguez-Talou J, Boland R (2003) Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures. Plant Sci 165:429–436

    Article  CAS  Google Scholar 

  • Wiktorowska E, Długosz M, Janiszowska W (2010) Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L. Enzyme Microb Technol 46:14–20. doi:10.1016/j.enzmictec.2009.09.002

    Article  CAS  Google Scholar 

  • Woelwer-Rieck U, Lankes C, Wawrzun A, Matthias W (2010) Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana. Eur Food Res Technol 231:581–588. doi:10.1007/s00217-010-1309-4

    Article  CAS  Google Scholar 

  • Xu S, Zhou W, Pottinger S, Baldwin IT (2015) Herbivore associated elicitor-induced defences are highly specific among closely related Nicotiana species. BMC Plant Biol 15(2):1–13. doi:10.1186/s12870-014-0406-0

    Google Scholar 

  • Yamazaki T, Flores HE (1991) Examination of steviol glucosides production by hairy root and shoot cultures of Stevia rebaudiana. J Nat Prod 54:986–992. doi:10.1021/np50076a010

    Article  CAS  Google Scholar 

  • Yue W, Ming QL, Lin B, Rahman K, Zheng CJ, Han T, Qin LP (2014) Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites. Crit Rev Biotechnol 36(2):215–232. doi:10.3109/07388551.2014.923986

    Article  PubMed  Google Scholar 

  • Zhang CH, Fevereiro PS, He G, Chen Z (2007) Enhanced paclitaxel productivity and release capacity of Taxus chinensis cell suspension cultures adapted to chitosan. Plant Sci 172(1):158–163. doi:10.1371/journal.pone.0104005

    Article  CAS  Google Scholar 

  • Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333. doi:10.1016/j.biotechadv.2005.01.003

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This research was financed by Ahi Evran University Scientific Research Projects Commission (PYO-MÜH.4001.14.006). All of the experiments were carried out at the Bioengineering Department of Ege University.

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Authors and Affiliations

  1. Genetic and Bioengineering Department, Faculty of Engineering and Architecture, Ahi Evran University, 40100, Kirsehir, Turkey

    Meltem Bayraktar, Esra Ilhan & Begum Akyol

  2. Biotechnology Department, Graduate School of Nature and Applied Sciences, Ege University, 35100, Bornova, Izmir, Turkey

    Elmira Naziri & Fatih Karabey

  3. Bioengineering Department, Faculty of Engineering, Ege University, 35100, Bornova, Izmir, Turkey

    Ismail Hakki Akgun, Erdal Bedir & Aynur Gurel

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  1. Meltem Bayraktar
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  2. Elmira Naziri
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Bayraktar, M., Naziri, E., Akgun, I.H. et al. Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana . Plant Cell Tiss Organ Cult 127, 289–300 (2016). https://doi.org/10.1007/s11240-016-1049-7

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