Skip to main content

Micropropagation for mass multiplication and enriched production of ascorbic acid in tissue culture foliage of roselle (Hibiscus sabdariffa L.)

In Vitro Cellular & Developmental Biology - Plant volume 52, pages 427–436 (2016)Cite this article

Abstract

An efficient protocol was developed for the micropropagation of Hibiscus sabdariffa through nodal explants (auxillary bud cultures) of in vitro seedlings. Nodal explants were able to induce multiple shoots on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg L−1 6-benzylaminopurine, 0.5 mg L−1 indole-3-acetic acid, and 10 μM silver nitrate (AgNO3; 7–8 shoots/explant) or 20 μg L−1 triacontanol (TRIA; 4–5 shoots/explant). Medium devoid of AgNO3 that was supplemented with 0.1–0.5 mg L−1 gibberellic acid (GA3) induced the proliferation of up to two shoots/explant, but supplementation with 1–5 mg L−1 GA3 induced a single shoot. The highest frequency of shoot elongation and rooting was obtained on 0.5-strength MS medium supplemented with 0.1 or 0.5 mg L−1 GA3, and on this medium, further proliferation of shoots was also evident. Micropropagated plants were hardened in the greenhouse and successfully established in soil. Ascorbic acid level in leaves of micropropagated plants from AgNO3- and TRIA-supplemented media was highest (2.24 ± 0.14 and 2.18 ± 0.13 mg g−1 fresh weight, respectively), which was 2.8-fold more than normal ex vitro leaves of the same age (0.79 ± 0.03 mg g−1 fresh weight).

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

Figure 1.
Figure 2.
Figure 3.

References

  • Addo AA (2004) Seasonal availability of dietary ascorbic acid and incidence of scurvy in northern state of Nigeria. Dissertation, Department of Biochemistry, ABU Zaria

  • Akitha Devi MK, Sakthivelu G, Giridhar P, Ravishankar GA (2012) Protocol for augmented shoot organogenesis in selected variety of [Glycine max L. (Merr.)]. Ind J Exp Biol 50:729–734

    Google Scholar 

  • Aloni R, Avni A, Dayan J (2007) Compositions for silencing the expressing the expression of gibberellin 2-oxidase and uses thereof. International application published under the patent cooperation treaty (PCT) WO/2007/135685, pp 54

  • Anonymous (1959) A dictionary of Indian raw materials and industrial products. Raw materials. Council of scientific and industrial Research, Delhi

    Google Scholar 

  • Ayadi R, Hamrouni L, Hanana M, Bouzid S, Trifi M, Khouja ML (2011) In vitro propagation and regeneration of an industrial plant kenaf (Hibiscus cannabinus L.). Ind Crop Prod 33:474–480

    CAS  Article  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    Google Scholar 

  • Biernbaum JA, Houtz RL, Ries SK (1988) Field studies with crops treated with colloidally dispersed triacontanol. J Am Soc HortSci 113:679–684

    CAS  Google Scholar 

  • Brouk B (1975) Plants consumed by men. Academic Press, London

    Google Scholar 

  • Chibnall AC, Williams EF, Latner AL, Piper SH (1933) The isolation of n-triacontanol from lucerne wax. Biochem J 27:1885–1888

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Da-Costa-Rocha I, Bonnlaender B, Sievers H, Pischel I, Heinrich M (2014) Hibiscus sabdariffa L.—a phytochemical and pharmacological review. Food Chem 165:424–443

    CAS  Article  PubMed  Google Scholar 

  • Davey MW, Montagu MV, Inze D, Sanmartin M, Kanellis A, Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J (2000) Review: plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric 80:825–860

    CAS  Article  Google Scholar 

  • Duhoky MSM, Rasheed KA (2010) Effect of different concentration of BA and IAA on micropropagation of Gardenia jasminoides. Mesopotamia J Agric 38:1–14

    Google Scholar 

  • Emmy HKI (2006) Chemical composition, antioxidant properties and hypocholesterolemic effects of differently treated roselle (Hibiscus sabdariffa L.) seeds. Dissertation, Universiti Putra Malaysia, Selangor, Malaysia

  • Giridhar P, Indu EP, Chandrasekar A, Ravishankar GA (2004) Influence of triacontanol on somatic embryogenesis in Coffea arabica L. And Coffea canephora p.Ex.Fr. In Vitro Cell Dev Biol Plant 40:200–203

    Article  Google Scholar 

  • Giridhar P, Reddy BO, Ravishankar GA (2001) Silver nitrate influences in vitro shoot multiplication and root formation in Vanilla planifolia. CurrSci 81:101–103

    Google Scholar 

  • Giridhar P, Sowmya KS, Ramakrishna A, Ravishankar GA (2010) Rapid clonal propagation and stevioside profiles of Stevia rebaudiana Bertoni. Int J Plant DevBiol 4:47–52

    Google Scholar 

  • Giridhar P, Vijayaramu D, Ravishankar GA (2003) Effect of silver nitrate on in vitro shoot growth of Coffea arabica and Coffea canephora. Trop Sci 43:144–146

    Article  Google Scholar 

  • Gomez-Leyva JF, Martinez-Acosta LA, Lopez-Muraira IG, Silos-Espino H, Ramirez-Cervantes F, Andrade-Gonzalez I (2008) Multiple shoot regeneration of roselle (Hibiscus sabdariffa L.) from a shoot apex culture system. Int J Bot 4:326–330

    Article  Google Scholar 

  • Govinden-Soulange J, Boodia N, Dussooa C, Gunowa R, Deensah S, Facknath S, Rajkomar B (2009) Vegetative propagation and tissue culture regeneration of Hibiscus sabdariffa L. (Roselle). World J AgrSci 5:651–661

    CAS  Google Scholar 

  • Habiba SU, Shimasaki K, Ahasan M, Alam M (2014) Effect of different cytokinins on in vitro organogenesis in protocorm-like bodies (PLBs) of Epidendrum ‘rouge star no. 8’. Middle East J Sci Res 21:1843–1847

    CAS  Google Scholar 

  • Herath SP, Suzuki T, Hattori K (2004) Multiple shoot regeneration from young shoots of kenaf (Hibiscus cannabinus). Plant Cell Tiss Organ Cult 77:49–53

    CAS  Article  Google Scholar 

  • Huda KMK, Bhuiyan MSR, Kabir MH, Jamal Uddin AFM (2007) In vitro plant regeneration protocol of Tossa jute (Corchorus olitorius). Int J Integr Biol 1:96–101

    CAS  Google Scholar 

  • Jain RK, Bal S (1997) Properties of pearl millet. J Agric Eng Res 66:85–91

    Article  Google Scholar 

  • Jenderek M, Olney R (2001) Hibiscus syriacus Plant regeneration from callus. Int Plant Prop Proc 50:565–568

    Google Scholar 

  • Khan MI, Sri Harsha PSC, Giridhar P, Ravishankar GA (2011) Pigment identification, antioxidant activity, and nutrient composition of Tinospora cordifolia (willd.) Miers ex Hook.F &Thoms fruit. Int J Food SciNutr 62:239–249

    CAS  Article  Google Scholar 

  • Khatun A, Naher Z (2002) Optimization of culture conditions for plant regeneration from explants of kenaf and mesta. Annual Report. Bangladesh Jute Res. Inst., Dhaka, Bangladesh, pp 57–58

  • Kumar SS, Manoj P, Giridhar P (2015b) Optimization of an in vitro protocol for the production of ascorbic acid in Hibiscus cannabinus leaf-derived normal root cultures. Eurasia J Biosci 9:38–45

    Google Scholar 

  • Kumar SS, Manoj P, Shetty NP, Giridhar P (2015a) Effect of different drying methods on chlorophyll, ascorbic acid and antioxidant compounds retention of leaves of Hibiscus sabdariffa L. J Sci Food Agric 95:1812–1820

    CAS  Article  PubMed  Google Scholar 

  • Kumar V, Giridhar P, Ravishankar GA (2009) AgNO3—a potential regulator of ethylene activity and plant growth modulator. Electron J Biotechnol 12:8–9

    Article  Google Scholar 

  • Kuriyan R, Kumar DR, Kurpad AV (2010) An evaluation of the hypolipidemic effect of an extract of Hibiscus sabdariffa leaves in hyperlipidemic Indians: a double blind, placebo controlled trial. BMC Comp Alt Med 10:27

    Article  Google Scholar 

  • Lichtenthaler HK (1996) Vegetation stress: an introduction to the stress concept in plants. J Plant Physiol 148:4–14

    CAS  Article  Google Scholar 

  • Lin HH, Chen JH, Wang CJ (2011) Chemopreventive properties and molecular mechanisms of the bioactive compounds in Hibiscus sabdariffa Linne. Current Med Chem 18:1245–1254

    CAS  Article  Google Scholar 

  • Mahadevan N, Shivali KP, Kamboj P (2009) Hibiscus sabdariffa Linn: an overview. Nat Prod Rad 8:77–83

    Google Scholar 

  • Mat Isa A, Isa PM, Abd Aziz AR (1985) Analisis kimia dan pemprosesan roselle (Hibiscus sabdariffa L.). Mardi Res Bull 13:68–74

    Google Scholar 

  • Mayol-Soto ML, Aragón-Vargas LF (2002) Post-exercise rehydration with different beverages: pure water, sports drink, and roselle tea. Pensar en Movimiento 2:41–54

    Article  Google Scholar 

  • Mohamed BB, Sulaiman AA, Dahab AA (2012) Roselle (Hibiscus sabdariffa L.) in Sudan, cultivation and their uses. Bull Env Pharmacol Life Sci 1:48–54

    Google Scholar 

  • Mohamed R, Fernandez J, Pineda M, Aguilar M (2007) Roselle (Hibiscus sabdariffa) seed oil is a rich source of gamma-tocopherol. J Food Sci 72:207–211

    Article  Google Scholar 

  • Morton JF (1987) Roselle. In: Fruits of warm climates. Miami, Florida, USA pp 281–286

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

    CAS  Article  Google Scholar 

  • Parimalan R, Giridhar P, Gururaj HB, Ravishankar GA (2009) Micropropagation of Bixa orellana using phytohormones and triacontanol. Biol Plant 53:347–350

    CAS  Article  Google Scholar 

  • Pau LT, Salmah Y, Suhaila M (2002) Antioxidative properties of roselle (Hibiscus sabdariffa L.) in linoleic acid model system. Nutr Food Sci 32:17–20

    Article  Google Scholar 

  • Purseglove JW (1968) Tropical crop dicotyledons 2. Longmans, Green and Co. Ltd., London,  pp 370–374

    Google Scholar 

  • Raoul SS, Gilbert C, Hamidou SF, Yannick T, Yao D, Abdourahamane S, Michle B (2010) Protocol for callus and somatic embryogenesis initiation for Hibiscus sabdariffa L. (Malvaceae): influence of explant type, sugar and plant growth regulator. Aus J Crop Sci 4:98–106

    Google Scholar 

  • Reddy BO, Giridhar P, Ravishankar GA (2001) In vitro rooting of Decalepis hamiltonii (Wight & Arn), an endangered shrub, by auxins and root promoting agents. Curr Sci 81:1479–1482

    CAS  Google Scholar 

  • Reddy BO, Giridhar P, Ravishankar GA (2002) The effect of triacontanol on micropropagation of Capsicum frutescens and Decalepis hamiltonii W&a. Plant Cell Tiss Organ Cult 71:253–258

    Article  Google Scholar 

  • Ries S, Houtz R (1983) Triacontanol as a plant growth regulator. HortSci 18:654–662

    CAS  Google Scholar 

  • Ries SK, Wert V, Sweeley CC, Leavitt RA (1977) Triacontanol: a new naturally occurring plant growth regulator. Science 195:1339–1341

    CAS  Article  PubMed  Google Scholar 

  • Sarkiyayi S, Ikioda H (2010) Estimation of thiamin and ascorbic acid contents in fresh and dried Hibiscus sabdariffa (roselle) and Lactuca sativa (lettuce). Adv J Food SciTechnol 2:47–49

    CAS  Google Scholar 

  • Sharma A, Kumar V, Giridhar P, Ravishankar GA (2008) Induction of in vitro flowering in Capsicum frutescens under the influence of silver nitrate and cobalt chloride and pollen. Electron J Biotechnol 11:1–6

    Article  Google Scholar 

  • Smirnoff N (2000) Ascorbic acid metabolism and functions of multi-faceted molecule. Curr Opin Plant Biol 3:229–235

    CAS  Article  PubMed  Google Scholar 

  • Sri Harsha PSC, Khan MI, Giridhar P, Ravishankar GA (2012) In vitro propagation of Rivina humilis L. Through proliferation of axillary shoots and shoot tips of mature plants. Ind J Biotech 11:481–485

    CAS  Google Scholar 

  • Srivatanakul M, Park SH, Sanders JR, Salas MG, Smith RH (2000) Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system. Plant Cell Rep 19:1165–1170

    CAS  Article  Google Scholar 

  • Tantos A, Meszaros A, Kissimon J (1999) The effect of triacontanol on micropropagation of balm, Melissa officinalis L. Plant Cell Rep 19:88–91

    CAS  Article  Google Scholar 

  • Vanderslice JT, Higgs DJ, Hayes JM, Block G (1990) Ascorbic acid and dehydroascorbic acid content of foods-as-eaten. J Food Comp Anal 3:105–118

    CAS  Article  Google Scholar 

  • Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393:365–369

    CAS  Article  PubMed  Google Scholar 

  • William CN, Chew WY, Rijaratuam JH (1980) Tree and field crops of the wetter regions of the tropics. Longman, London, pp. 64–72

    Google Scholar 

  • Zapata C, Srivatanakul M, Park SH, Lee BM, Salas MG, Smith RH (1999) Improvements in shoot apex regeneration of two fiber crops: cotton and kenaf. Plant Cell Tiss Organ Cult 56:185–191

    Article  Google Scholar 

Download references

Acknowledgments

The authors are thankful to the Department of Biotechnology, Government of India, New Delhi, for the financial assistance (BT/PR1238/FNS/20/524/2011). We gratefully acknowledge the Director, CSIR-CFTRI, for his kind support.

Author information

Authors and Affiliations

  1. Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore, 570 020, India

    Sandopu Sravan Kumar, Prabhakaran Manoj & Parvatam Giridhar

Authors
  1. Sandopu Sravan Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Prabhakaran Manoj
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Parvatam Giridhar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Parvatam Giridhar.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Editor: Praveen K. Saxena

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kumar, S.S., Manoj, P. & Giridhar, P. Micropropagation for mass multiplication and enriched production of ascorbic acid in tissue culture foliage of roselle (Hibiscus sabdariffa L.). In Vitro Cell.Dev.Biol.-Plant 52, 427–436 (2016). https://doi.org/10.1007/s11627-016-9785-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-016-9785-2

Keywords

  • Micropropagation
  • Nodal explants
  • Multiple shoots
  • In vitro rooting
  • Silver nitrate
  • Triacontanol