Skip to main content
SpringerLink
Log in

The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value

  • Research Article
  • Published:
Physiology and Molecular Biology of Plants Aims and scope Submit manuscript

Abstract

An improved micropropagation protocol facilitating continuous multiplication of elite germplasm of Moringa oleifera has been developed. Initial culture of nodal explant in MS medium supplemented with 2.5 µM BA resulted in the formation of 12.5 shoots per explant with high frequency of leaf fall (84.3%). To confirm whether the leaf fall is due to accumulation of ethylene in the culture vessel, effect of ethylene releasing agent CEPA in the medium was tested. In order to reduce leaf fall and improve multiplication, varying concentration of anti-ethylene agent, AgNO3 was incorporated in the medium. Addition of 2.5 μM AgNO3 in combination with 2.5 μM BA produced maximum number of shoots (17.6) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots, where significant reduction in leaf fall (20.6%) was noticed. This enabled sustained multiplication of M. oleifera through continuous subculture without adversely affecting shoot number or shoot quality in terms of shoot length. Microshoots obtained from fourth subculture onwards were used for ex vitro rooting and found that by treating 50 µM NAA for 30 s, maximum numbers of microshoots (83.3%) were rooted. Rooted plants were acclimatized, survived and were successfully transferred to field. Genetic fidelity analysis using 10 ISSR primers revealed more than 95% monomorphic bands among plants raised in MS medium containing low concentration (2.5 µM) of AgNO3 and BA (2.5 µM). The addition of AgNO3 in the medium sustained in vitro growth and effectively prevented leaf fall compared to control, thus demonstrating efficient micropropagation of M. oleifera.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Abeles FB (1973) Ethylene in plant biology. Academic Press, New York

    Google Scholar 

  • Agarwal A, Purwar JP (2013) Influence of silver nitrate on in vitro performance of microplants of Potato Cultivar ‘Kufri Himalini’. Vegetos Int J Plant Res 26:96–100. https://doi.org/10.5958/j.2229-4473.26.1.014

    Article  Google Scholar 

  • Al Khateeb W, Bahar E, Lahham J, Schroeder D, Hussein E (2013) Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR). Physiol Mol Biol Plants 19:157–164. https://doi.org/10.1007/s12298-012-0149-z

    Article  CAS  PubMed  Google Scholar 

  • Alva Ticona S, Oropeza M (2013) Effect of culture medium consistence and silver nitrate on micropropagation of two potato (Solanum tuberosum) cultivars. Revista Colombiana de Biotecnologia 15:55–62

    Article  Google Scholar 

  • Avila-Trevino JA, Muñoz-Alemán JM, Perez-Molphe-Balch E, Rodríguez-Sahagun A, Morales-Domínguez JF (2017) In vitro propagation from bud and apex explants of Moringa oleifera and evaluation of the genetic stability with RAMP marker. South Afr J Bot 108:149–156. https://doi.org/10.1016/j.sajb.2016.10.003

    Article  CAS  Google Scholar 

  • Barche S, Sharma KSKA, Mishra PK (2013) Standardization of propagation method in drumstick cv. PKM-1. Nat Sci 11:141–143

    Google Scholar 

  • Beyer EM (1975) Abscission: the initial effect of ethylene is in the leaf blade. Plant Physiol 55:322–327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Beyer EM (1976a) A potent inhibitor of ethylene action in plants. Plant Physiol 58:268–271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Beyer EM (1976b) Silver ion: a potent anti-ethylene agent in cucumber and tomato. HortScience 11:175–196

    Google Scholar 

  • Beyer EM (1979) [14C] Ethylene metabolism during leaf abscission in cotton. Plant Physiol 64:971–974. https://doi.org/10.1104/pp.64.6.971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bleecker AB, Kende H (2000) Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Dev Biol 6:1–18

    Article  Google Scholar 

  • Burg SP (1968) Ethylene, plant senescence and abscission. Plant Physiol 43:1503–1511

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chandrika M, Rai VR (2010) ISSR marker based analysis of micropropagated plantlets of Nothapodytes foetida. Biol Plant 54:561–565

    Article  CAS  Google Scholar 

  • Chandrika M, Rai VR, Kini KR (2008) Assessment of genetic stability of in vitro grown Dictyospermum ovalifolium. Biol Plant 52:735–739

    Article  CAS  Google Scholar 

  • Chraibi BKM, Latche A, Raustan JP, Fallot J (1991) Stimulation of shoot regeneration from cotyledons of Helianthus annuus by ethylene inhibitors silver and cobalt. Plant Cell Rep 10:204–207

    Google Scholar 

  • Cristea TO, Leonte C, Brezeanu C, Brezeanu M, Ambarus S, Calin M, Prisecaru M (2012) Effect of AgNO3 on androgenesis of Brassica oleracea L. anthers cultivated in vitro. Afr J Biotechnol 11:13788–13795

    CAS  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–22

    Article  CAS  Google Scholar 

  • Forster N, Mewis I, Ulrichs C (2013) Moringa oleifera-establishment and multiplication of different ecotypes in vitro. Gesunde Pflanzen 65(1):21–31. https://doi.org/10.1007/s10343-013-0291-8

    Article  Google Scholar 

  • Gayathri M, Kumar PS, Prabha AML, Muralitharan G (2015) In vitro regeneration of Arachis hypogaea L. and Moringa oleifera Lam. using extracellular phytohormones from Aphanothece sp. MBDU 515. Algal Res 7:100–105. https://doi.org/10.1016/j.algal.2014.12.009

    Article  Google Scholar 

  • Gonzalez A, Arigita L, Majada J, Tames RS (1997) Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L. Plant Growth Regul 22:1–6

    Article  CAS  Google Scholar 

  • Hassanein AM, Salem JM, Faheed FA, El-Nagish A (2018) Effect of anti-ethylene compounds on isoenzyme patterns and genome stability during long term culture of Moringa oleifera. Plant Cell Tissue Organ Cult 132:201–212. https://doi.org/10.1007/s11240-017-13260

    Article  CAS  Google Scholar 

  • Islam S, Jahan MAA, Khatun R (2005) In vitro regeneration and multiplication of year-round fruit bearing Moringa oleifera L. J Biol Sci 5:145–148

    Article  Google Scholar 

  • Johnson PR, Ecker JR (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annu Rev Genet 32:227–254

    Article  CAS  PubMed  Google Scholar 

  • Jun-jie Z, Yue-sheng Y, Meng-fei L, Shu-qi L, Yi T, Han-bin C, Xiao-yang C (2017) An efficient micropropagation protocol for direct organogenesis from leaf explants of an economically valuable plant, drumstick (Moringa oleifera Lam.). Ind Crops Prod 103:59–63. https://doi.org/10.1016/j.indcrop.2017.03.028

    Article  CAS  Google Scholar 

  • Kantharajah AS, Dodd WA (1991) Rapid clonal propagation of Moringa oleifera Lam., using tissue culture. South Indian Hortic 39:224–228

    Google Scholar 

  • Kao CH, Yang SF (1983) Role of ethylene in the senescence of detached rice leaves. Plant Physiol 73:881–885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lemos EEP, Jennet B (2015) Control of leaf abscission in nodal cultures of Annona squamosa L. J Hortic Sci Biotechnol 71:721–728

    Article  Google Scholar 

  • Marfori EC (2011) Clonal micropropagation of Moringa oleifera L. Philipp Agric Sci 93:454–457

    Google Scholar 

  • Mathur M, Yadav S, Katariya PK, Kamal R (2014) In vitro propagation and biosynthesis of steroidal sapogenins from various morphogenetic stages of Moringa oleifera Lam., and their antioxidant potential. Acta Physiol Plant 36:1749–1762. https://doi.org/10.1007/s11738-014-1549-1

    Article  CAS  Google Scholar 

  • Mehta A, Agrawal B (2008) Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity. Orient Pharm Exp Med 8:24–31. https://doi.org/10.3742/OPEM.2008.8.1.024

    Article  Google Scholar 

  • Mirzai F, Uliaie ED, Hagh AB (2015) Stimulation effect of AgNO3 and CoCl2 as ethylene inhibitors on in vitro organogenesis of sunflower (Helianthus annuus L.). J Agric Sci 25:113–118

    Google Scholar 

  • Mookkan M, Andy G (2014) AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper. Plant Signal Behav 9(10):1–5. https://doi.org/10.4161/psb.32165

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Naik SK, Chand PK (2003) Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.). J Plant Physiol 160:423–430

    Article  CAS  PubMed  Google Scholar 

  • Obata-Sasamoto H, Nishi N, Komamine A (1981) Mechanism of suppression of DOPA accumulation in a callus culture of Stizolobium hassjoo. Plant Cell Physiol 22:827–835

    CAS  Google Scholar 

  • Prakash PK, Prakash L, Trevor AT (1998) Review: regulation of morphogenesis in plant tissue culture by ethylene. Vitro Cell Dev Biol Plant 34:94–103

    Article  Google Scholar 

  • Pua EC, Chi GL (1993) De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiol Plant 88:467–474

    Article  CAS  Google Scholar 

  • Ramachandran C, Peter KV, Gopalakrishnan PK (1980) Drumstick (Moringa oleifera) a multipurpose Indian vegetable. Econ Bot 34:276–283

    Article  CAS  Google Scholar 

  • Riyathong T, Dheeranupattana S, Palee J, Shank L (2010) Shoot multiplication and plant regeneration from in vitro cultures of drumstick tree (Moringa oleifera Lam.). In: The 8th international symposium on biocontrol and biotechnology. King Mongkut’s Institute of Technology Ladkrabang and Khon Kaen University, Nongkhai Campus, Thailand, pp 99–104

  • Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB (1999) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283:996–998

    Article  CAS  PubMed  Google Scholar 

  • Saini RK, Shetty NP, Giridhar P, Ravishankar GA (2012) Rapid in vitro regeneration method for Moringa oleifera and performance evaluation of field grown nutritionally enriched tissue cultured plants. 3 Biotech 2:187–192. https://doi.org/10.1007/s13205-012-0045-9

    Article  PubMed Central  Google Scholar 

  • Saini RK, Saad KR, Ravishankar GA, Giridhar P, Shetty NP (2013) Genetic diversity of commercially grown Moringa oleifera Lam. cultivars from India by RAPD, ISSR and cytochrome P450-based markers. Plant Syst Evolut 299:1205–1213. https://doi.org/10.1007/s00606-013-0789-7

    Article  CAS  Google Scholar 

  • Saini RK, Manoj P, Shetty NP, Srinivasan K, Giridhar P (2014a) Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats. Nutr Res 34:630–638

    Article  CAS  PubMed  Google Scholar 

  • Saini RK, Shetty NP, Giridhar P (2014b) Carotenoid content in vegetative and reproductive parts of commercially grown Moringa oleifera Lam. cultivars from India by LC–APCI–MS. Eur Food Res Technol 238:971–978

    Article  CAS  Google Scholar 

  • Saini RK, Manoj P, Shetty NP, Srinivasan K, Giridhar P (2016) Relative bioavailability of folate from the traditional food plant Moringa oleifera L. as evaluated in a rat model. J Food Sci Technol 53:511–520

    Article  CAS  PubMed  Google Scholar 

  • Salem JM (2016) In vitro propagation of Moringa oleifera L. under salinity and ventilation conditions. Genet Plant Physiol 6:54–64

    Google Scholar 

  • Sarropoulou V, Dimassi-Theriou K, Therios I (2016) Effect of the ethylene inhibitors silver nitrate, silver sulfate, and cobalt chloride on micropropagation and biochemical parameters in the cherry rootstocks CAB-6P and Gisela 6. Turk J Biol 40:670–683. https://doi.org/10.3906/biy-1505-92

    Article  CAS  Google Scholar 

  • Shokoohmand A, Drew RA (2013) Micropropagation of Moringa oleifera. Acta Hort 988:149–160. https://doi.org/10.17660/ActaHortic.2013.988.17

    Article  Google Scholar 

  • Sirisom Y, Te-chato S (2012) The effect of peptone and silver nitrate on in vitro shoot formation in Hevea brasiliensis Muell Arg. J Agric Technol 8:1509–1516

    Google Scholar 

  • Sisler EC, Yang SF (1984) Ethylene the gaseous plant hormone. Bioscience 34:234–238

    Article  CAS  Google Scholar 

  • Sreeranjini S, Siril EA (2014) Field performance and genetic fidelity evaluation of micropropagated Morinda citrifolia L. Indian J Biotechnol 13:121–130

    CAS  Google Scholar 

  • Steinitz B, Tabib Y, Gaba V, Gefen T, Vaknin Y (2009) Vegetative micro-cloning to sustain biodiversity of threatened Moringa species. Vitro Cell Dev Biol Plant 45:65–71. https://doi.org/10.1007/s11627-008-9162-x

    Article  Google Scholar 

  • Stephenson KK, Fahey JW (2004) Development of tissue culture methods for the rescue and propagation of endangered Moringa spp. germplasm. Econ Bot 58:116–124

    Article  Google Scholar 

  • Sung LS, Huang SY (2000) Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3, 4-dihydroxyphenylalanine. Biotechnol Lett 22:875–878

    Article  CAS  Google Scholar 

  • Tamimi SM (2015) Effects of ethylene inhibitors, silver nitrate (AgNO3), cobalt chloride (CoCl2) and aminooxy acetic acid (AOA), on in vitro shoot induction and rooting of banana (Musa acuminata L.). Afr J Biotechnol 14:2511–2516. https://doi.org/10.5897/AJB2015.14788

    Article  Google Scholar 

  • Thorpe TA (2007) History of plant tissue culture. Mol Biotechnol 37:169–180. https://doi.org/10.1007/s12033-007-0031-3

    Article  CAS  PubMed  Google Scholar 

  • Tiwari SK, Tiwari KP, Siril EA (2002) An improved micropropagation protocol for teak. Plant Cell Tissue Organ Cult 71:1–6

    Article  CAS  Google Scholar 

  • Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007) Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers. Vitro Cell Dev Biol Plant 43:267–274. https://doi.org/10.1007/s1627-007-9028-7

    Article  CAS  Google Scholar 

  • Vinod K, Giridhar P, Ravishankar GA (2009) Review: AgNO3—a potential regulator of ethylene activity and plant growth modulator. Electron J Biotechnol 12(2):1–15

    Google Scholar 

  • Wang KLC, Hai L, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14:31–51

    Article  CAS  Google Scholar 

  • Yan H, Liang C, Yang L, Li Y (2010) In vitro and ex vitro rooting of Siratia grosvenorii, a traditional medicinal plant. Acta Physiol Plant 32:115–120. https://doi.org/10.1007/s11738-009-0386-0

    Article  CAS  Google Scholar 

  • Zhao XC, Qu X, Mathews DE, Schaller GE (2002) Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiol 130:1983–1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. Suhara Beevy S, Professor and Head, Department of Botany, University of Kerala for the facilities provided. DRS wish to thank University of Kerala for granting fellowship (No. Ac. EI/A2/10625/2016-I) to undertake the present work.

Author information

Authors and Affiliations

  1. Department of Biotechnology, University of Kerala, Kariavattom, Thiruvananthapuram, 695 581, India

    R. S. Drisya Ravi

  2. Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram, 695 581, India

    E. A. Siril & Bindu R. Nair

Authors
  1. R. S. Drisya Ravi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. A. Siril
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bindu R. Nair
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

DRS conducted the experiments. DRS and EAS analyzed the data. DRS drafted the manuscript. EAS and BRN designed the experiments. EAS, BRN and DRS revised the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to E. A. Siril.

Ethics declarations

Conflict of interest

All authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Drisya Ravi, R.S., Siril, E.A. & Nair, B.R. The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value. Physiol Mol Biol Plants 25, 1311–1322 (2019). https://doi.org/10.1007/s12298-019-00689-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-019-00689-x

Keywords

Search

Navigation