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Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors

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Abstract

An efficient regeneration protocol for the medicinal plant, Paris polyphylla Sm. was developed through the formation of mini-rhizomes (MRs) using transverse thin cell layer (tTCL) culture technique. MRs were induced from tTCL explants derived from the basal and middle stem portions while apical portion failed to show any kind of response. Highest response percentage (86.6 %) of MRs formation with a maximum fresh weight (1.05 ± 0.08 g) was achieved from basal sections cultured on ½ MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (BAP). MRs transferred to plant growth regulator free medium gave rise to shoot buds that eventually regenerated into plantlets and were successfully acclimatized with a survival percentage of more than 95 % under greenhouse conditions. Quantification through reverse-phase HPLC showed 1.41-fold higher content of total steroidal saponins in MRs cultured on medium supplemented with 0.5 mg/l BAP as compared to the field-grown rhizome. Elicitation of MRs liquid culture with chitosan, salicyclic acid (SA) and yeast extract enhanced the production of steroidal saponins but resulted in reduced growth rate. Highest total steroidal saponins content (87.66 ± 1.66 mg/g DW) was achieved in cultures treated with SA at 50 mg/l after 30 days of elicitation which is 3.6 times higher than the in vivo rhizome. The developed protocol would facilitate the conservation of this valuable medicinal plant and could be used as a ready stock to meet the demands of the pharmaceutical industry for steroidal saponins productions.

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Abbreviations

BAP:

6-Benzylaminopurine

CHI:

Chitosan

DW:

Dry weight

FW:

Fresh weight

HPLC:

High performance liquid chromatography

KIN:

Kinetin

MR:

Mini-rhizome

MS:

Murashige and Skoog

SA:

Salicyclic acid

TDZ:

Thidiazuron

tTCL:

Transverse thin cell layer

PGR:

Plant growth regulator

YE:

Yeast extract

References

  • Aitsu Y, Yokoyama M, Yanagi M (1992) Production of ginsenosides by culture of transformed ginseng. Jpn Kokai Tokkyo Koho JPO4341194

  • Ali MB, Yu KW, Hahn EJ, Paek KY (2006) Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors. Plant Cell Rep 25:613–620

    Article  CAS  PubMed  Google Scholar 

  • Baskaran P, Jayabalan N (2008) Effect of growth regulators on rapid micropropagation and psoralen production in Psoralea corylifolia L. Acta Physiol Plant 30:345–351

    Article  CAS  Google Scholar 

  • Bhattarai KR, Ghimire MD (2006) Cultivation and sustainable harvesting of commercially important medicinal and aromatic plants of Nepal. Heritage Research and Development Forum, Nepal, pp 369–372

    Google Scholar 

  • Cheng XY, Zhou HY, Cui X, Ni W, Liu CZ (2006) Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor. J Biotechnol 121:253–260

    Article  CAS  PubMed  Google Scholar 

  • Cheung JYN, Ong RCY, Suen YK, Ooi V, Wong HNC et al (2005) Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. Cancer Lett 217:203–211

    Article  PubMed  Google Scholar 

  • Cho JS, Kim JY, Kim IH, Kim DI (2003) Effect of polysaccharide elicitors on the production of decursinol angelate in Angelica gigas Nakai root cultures. Biotechnol Bioproc Eng 8:158–161

    Article  CAS  Google Scholar 

  • Costa F, Yendo ACA, Fleck JD, Gosmann G, Fett-Neto AG (2013) Accumulation of a bioactive triterpene saponin fraction of Quillaja brasiliensis leaves is associated with abiotic and biotic stresses. Plant Physiol Biochem 66:56–62

    Article  PubMed  Google Scholar 

  • Cousins MM, Adelberg JW (2008) Short-term and long-term time course studies of turmeric (Curcuma longa L.) microrhizome development in vitro. Plant Cell Tissue Organ Cult 93:283–293

    Article  Google Scholar 

  • Deng DW, Lauren DR, Cooney JM, Jensen DJ, Wurms KV, Upritchard JE, Cannon RD, Wang MZ, Li MZ (2008) Antifungal saponins from Paris polyphylla Smith. Planta Med 74:1397–1402

    Article  CAS  PubMed  Google Scholar 

  • Devkota KP, Tareq HKM, Ranjit R, Lannang AM, Samreen, Choudhary MI (2007) Tyrosinase inhibitory and antileishmanial constituents from the rhizomes of Paris polyphylla. Nat Prod Res 21:321–327

    Article  CAS  PubMed  Google Scholar 

  • Fernie AR, Willmitzer L (2001) Update on tuber formation, dormancy and sprouting: molecular and biochemical triggers of potato tuber development. Plant Physiol 127:1459–1465

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fu YL, Yu ZY, Tang XM, Zhao Y, Yuan XL, Wang S, Ma BP, Cong YW (2008) Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism. J Thromb Haemost 6:524–533

    Article  CAS  PubMed  Google Scholar 

  • Ghosh S, Ghosh B, Jha S (2007) In vitro tuberisation of Gloriosa superba L. on basal medium. Sci Hortic 114:220–223

    Article  CAS  Google Scholar 

  • Gopal J, Minocha JL, Dhaliwal HS (1998) Microtuberization in potato (Solanum tuberosum L.). Plant Cell Rep 17:794–798

    Article  CAS  Google Scholar 

  • Hahn MG (1996) Microbial elicitors and their receptors in plants. Annu Rev Phytopathol 34:387–412

    Article  CAS  PubMed  Google Scholar 

  • Han BH, Yae BW, Yu HJ, Peak KY (2005) Improvement of in vitro micropropagation of Lilium oriental hybrid ‘Casablanca’ by the formation of shoots with abnormally swollen basal plates. Sci Hortic 103:351–359

    Article  CAS  Google Scholar 

  • Hanafy MS, Abou-Setta LM (2007) Saponins production in shoot and callus cultures of Gypsophila paniculata. J Appl Sci Res 3:1045–1049

    CAS  Google Scholar 

  • He J, Zhang S, Wang H, Chen CX, Chen SF (2006) Advances in studies on and uses of Paris polyphylla var. yunnanensis. Acta Botanica Yunnanica 28:271–276

    CAS  Google Scholar 

  • Jean M, Cappadocia M (1991) In vitro tuberization in Dioscorea alata L. ‘Brazo fuerte and ‘Florida’ and D. abyssinica Hoch. Plant Cell Tissue Organ Cult 26:147–152

    Google Scholar 

  • Jeong GT, Park DH (2005) Enhancement of growth and secondary metabolite biosynthesis: effect of elicitors derived from plants and insects. Biotechnol Bioprocess Eng 10:73–77

    Article  CAS  Google Scholar 

  • Kang SM, Jung HY, Kang YM, Yun DJ, Bahk JD, Yang JK, Choi MS (2004) Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Sci 166:745–751

    Article  CAS  Google Scholar 

  • Karwasara VS, Jain R, Tomar P, Dixit VK (2010) Elicitation as yield enhancement strategy for glycyrrhizin production by cell cultures of Abrus precatorius Linn. In vitro Cell Dev Biol Plant 46:354–462

    Article  CAS  Google Scholar 

  • Kehie M, Kumaria S, Tandon P (2013) In vitro plantlet regeneration from cotyledon segments of Capsicum chinense Jacq. cv. Naga King Chili, and determination of capsaicin content by high performance liquid chromatography. Sci Hortic 164:1–8

    Article  CAS  Google Scholar 

  • Khare CP (2007) Indian medicinal plants—an illustrated dictionary. Springer, New Delhi, pp 16, 463–464

  • Korsangruang S, Soonthornchareonnon N, Chintapakorn Y, Saralamp P, Prathanturarug S (2010) Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria. candollei var. candollei and P. candollei var. mirifica cell suspension cultures. Plant Cell Tissue Organ Cult 103:333–342

    Article  CAS  Google Scholar 

  • Lee H, Lin JY (1988) Antimutagenic activity of extracts from anticancer drugs in Chinese medicine. Mutat Res 204:229–234

    Article  CAS  PubMed  Google Scholar 

  • Lee MS, Yuet-Wa JC, Kong SK, Yu B, Eng-Choon VO, Nai-Ching HW, Chung-Wai TM, Fung KP (2005) Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft. Cancer Biol Ther 4:1248–1254

    Article  CAS  PubMed  Google Scholar 

  • Li YC (1986) Vegetative propagation of Paris polyphylla var. yunnanensis. Acta Bot Yunnan 8:429–435 (in Chinese)

    Google Scholar 

  • Lipsky AK (1992) Problems of optimization of plant cell culture processes. J Biotechnol 26:83–97

    Article  PubMed  Google Scholar 

  • Ludwig-Müller J, Georgiev M, Bley T (2008) Metabolite and hormonal status of hairy root cultures of Devil’s claw (Harpagophytum procumbens) in flasks and in a bubble column bioreactor. Process Biochem 43:15–23

    Article  Google Scholar 

  • Man SL, Wang YL, Li YY, Gao WY, Huang XX, Ma CY (2013) Phytochemistry, pharmacology, toxicology and structure-cytotoxicity relationship of Paridis Rhizome saponin. Chin Herbal Med 5:33–46

    Google Scholar 

  • Mao AA, Hynniewta TM, Sanjappa M (2009) Plant wealth of northeast India with reference to ethnobotany. Indian J Tradit Knowl 8:96–103

    Google Scholar 

  • Mata-Rosas M, Baltazar-Garcıa RJ, Moon P, Hietz P, Luna-Monterrojo VE (2010) In vitro regeneration of Lycaste aromatica (Graham ex Hook) Lindl. (Orchidaceae) from pseudobulb sections. Plant Biotechnol Rep 4:157–163

    Article  Google Scholar 

  • Matsuda H, Pongpiriyadacha Y, Morikawa T, Kishi A, Kataoka S, Yoshikawa M (2003) Protective effects of steroid saponins from Paris polyphylla var. yunnanensis on ethanol- or indomethacin-induced gastric mucosal lesions in rats: structural requirement for activity and mode of action. Bioorg Med Chem Lett 13:1101–1106

    Article  CAS  PubMed  Google Scholar 

  • Merkli A, Christen P, Kapetanidis I (1997) Production of diosgenin by hairy root cultures of Trigonella foenum-graecum L. Plant Cell Rep 16:632–636

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nhut DT, Le BV, Fukai S, Tanaka M, Van TT (2001) Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem. Plant Growth Regul 33:59–65

    Article  Google Scholar 

  • Nhut DT, Teixeira da Silva JA, Le BV, Van Tran Thanh K (2003) Thin cell layer studies of vegetable, leguminous and medicinal plants. In: Nhut DT, Van Tran Thanh K, Le BV, Thorpe T (eds) Thin cell layer culture system: regeneration and transformation applications. Kluwer, Dordrecht, pp 387–426

    Chapter  Google Scholar 

  • Omokolo ND, Boudjeko T, Tsafack Takadong JJ (2003) In vitro tuberization of Xanthosoma sagittifolium L. Schott: effects of phytohormones, sucrose, nitrogen and photoperiod. Sci Hortic (Amsterdam) 98:337–345

    Article  CAS  Google Scholar 

  • Pan Y, Liang Y, Wang H, Liang M (2004) Antioxidant activities of several Chinese medicine herbs. Food Chem 88:347–350

    Article  CAS  Google Scholar 

  • Pence VC, Soukup VG (1993) Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro. Plant Cell Tissue Organ Cult 35:229–235

    Article  Google Scholar 

  • Piao XC, Chakrabarty D, Hahn EJ, Paek KY (2003) A simple method for mass production of potato microtubers using a bioreactor system. Curr Sci 84:1129–1132

    Google Scholar 

  • Poornima GN, Ravishankar RV (2007) In vitro propagation of wild yams, Dioscorea oppositifolia (Linn) and Dioscorea pentaphylla (Linn). Afr J Biotechnol 6:2348–2352

    CAS  Google Scholar 

  • Qian ZG, Zhao ZJ, Xu Y, Qian X, Zhong JJ (2006) Novel chemically synthesized salicylate derivative as an effective elicitor for inducing the biosynthesis of plant secondary metabolites. Biotechnol Prog 22:331–333

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Raomai S, Kumaria S, Tandon P (2014) Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-014-0496-2

  • Roat C, Ramawat KG (2009) Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin. Plant Biotechnol Rep 3:135–138

    Article  Google Scholar 

  • Sarkar D, Pandey SK, Sharma S (2006) Cytokinins antagonize the jasmonates action on the regulation of potato (Solanum tuberosum) tuber formation in vitro. Plant Cell Tissue Organ Cult 87:285–295

    Article  CAS  Google Scholar 

  • Scherwinski-Pereira JE, da Guedes RS, Fermino PCP Jr, Silva TL, Costa FHS (2010) Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique. In Vitro Cell Dev Biol-Plant 46:378–385

    Article  CAS  Google Scholar 

  • Sharma TR, Singh BM (1995) In vitro microrhizome production in Zingiber officinale Rosc. Plant Cell Rep 15:274–277

    CAS  PubMed  Google Scholar 

  • Shirgurkar MV, John CK, Nadgauda RS (2001) Factors affecting in vitro microrhizome production in turmeric. Plant Cell Tissue Organ Cult 64:5–11

    Article  CAS  Google Scholar 

  • Silva JAT (2003) Thin cell layer technology in ornamental plant micropropagation and biotechnology. Afr J Biotech 2:683–691

    Google Scholar 

  • Singh SK, Rai MK, Asthana P, Sahoo L (2009) An improved micropropagation of Spilanthes acmella L. through transverse thin cell layer culture. Acta Physiol Plant 31:693–698

    Article  CAS  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696

    Article  CAS  PubMed  Google Scholar 

  • Sudha G, Ravishankar GA (2002) Involvement and interaction of various signalling compounds on the plant metabolic events during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects. Plant Cell Tissue Organ Cult 71:181–212

    Article  CAS  Google Scholar 

  • Sun J, Liu BR, Hu WJ, Yu LX, Qian XP (2007) In vitro anticancer activity of aqueous extracts and ethanol extracts of fifteen traditional Chinese medicines on human digestive tumor cell lines. Phytother Res 21:1102–1104

    Article  CAS  PubMed  Google Scholar 

  • Suri SS, Jain S, Ramawat KG (1999) Plantlet regeneration and bulbil formation in vitro from leaf and stem explants of Curculigo orchioides, an endangered medicinal plant. Sci Hortic 79:127–134

    Article  CAS  Google Scholar 

  • Takayama S, Misawa M (1982) Regulation of organ formation by cytokinin and auxin in Lilium bulb scales grown in vitro. Plant Cell Physiol 22:461–467

    Google Scholar 

  • Tyagi RK, Agrawal A, Yusuf A (2006) Conservation of Zingiber germplasm through in vitro rhizome formation. Sci Hortic 108:210–219

    Article  CAS  Google Scholar 

  • Van Tran Thanh K (1974) Direct flower neoformation from superficial tissue of small explants of Nicotiana tabacum L. Planta 115:87–90

    Google Scholar 

  • Veerashree V, Anuradha CM, Kumar V (2012) Elicitor-enhanced production of gymnemic acid in cell suspension cultures of Gymnema sylvestre R. Br. Plant Cell Tissue Organ Cult 108:27–35

    Article  CAS  Google Scholar 

  • Vinterhalter B, Jankovic T, Sabikin K, Nicolic R, Vinterhalter D (2008) Propagation and xanthone content of Gentianella austriaca shoot cultures. Plant Cell Tissue Organ Cult 94:329–335

    Article  Google Scholar 

  • Wang GX, Han J, Zhao LW, Jiang DX, Liu YT, Liu XL (2010) Anthelmintic activity of steroidal saponins from Paris polyphylla. Phytomedicine 17:1102–1105

    Article  CAS  PubMed  Google Scholar 

  • Wink M (1989) Genes of secondary metabolism: differential expression in plants and in vitro cultures and functional expression in genetically transformed microorganisms. In: Kurz WGW (ed) Primary and secondary metabolism of plant cell cultures I. Springer, Berlin, pp 239–251

    Chapter  Google Scholar 

  • Wu SS, Gao WY, Duan HQ, Jia W (2004) Advances in studies on chemical constituents and pharmacological activities of Rhizoma paridis. Chin Tradit Herbal Drugs 35:344–347

    CAS  Google Scholar 

  • Zaerr JB, Mapes MO (1982) Action of growth regulators. In: Bonga JM, Durzan DJ (eds) Tissue culture in forestry. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague/Boston/London, pp 231–255

    Chapter  Google Scholar 

  • Zhang CH, Wu JY, He GY (2002) Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures. Appl Microbiol Biotechnol 60:396–402

    Article  CAS  PubMed  Google Scholar 

  • Zhang X, Cui Y, Huang J, Zhang Y, Nie Z et al (2007) Immuno-stimulating properties of diosgenyl saponins isolated from Paris polyphylla. Bioorg Med Chem Lett 17:2408–2413

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Zhao Y, Kang LP, Liu YX, Liang YG, Tan DW, Yu ZY (2009) Steroidal saponins from the rhizome of Paris polyphylla and their cytotoxic activities. Planta Med 75:356–363

    Article  CAS  PubMed  Google Scholar 

  • Zhao JL, Zhou LG, Wu JY (2010) Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Appl Microbiol Biotechnol 87:137–144

    Article  CAS  PubMed  Google Scholar 

  • Zhou J (1989) Some bioactive substances from plants of West China. Pure Appl Chem 61:457–460

    Article  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge Dr. A. Bhattacharjee and Ms. B. J. Mylliemngap, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong for providing the HPLC facilities and valuable help. The authors would also like to thank Prof. N. Venugopal, Department of Botany, North-Eastern Hill University, Shillong for permission to use microtome. SR is thankful to University Grant Commission, India for awarding her Rajiv Gandhi National Fellowship for SC/ST.

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Raomai, S., Kumaria, S., Kehie, M. et al. Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors. Plant Growth Regul 75, 341–353 (2015). https://doi.org/10.1007/s10725-014-9957-1

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