Skip to main content

In vitro propagation, micromorphological studies and ex vitro rooting of cannon ball tree (Couroupita guianensis aubl.): a multipurpose threatened species

Physiology and Molecular Biology of Plants volume 22, pages 131–142 (2016)Cite this article

Abstract

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l−1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l−1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l−1 each of BAP and Kinetin (Kin) + 0.5 mg l−1 α-naphthalene acetic acid (NAA) with additives (50 mg l−1 of ascorbic acid and 25 mg l−1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l−1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l−1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

AC:

Activated charcoal

BAP:

6-benzylaminopurine

IAA:

indole-3 acetic acid

IBA:

indole-3 butyric acid

Kin:

Kinetin

MS:

Murashige and Skoog’s (1962) medium

NAA:

α-naphthalene acetic acid

SPFD:

Spectral photon flux density

PGRs:

Plant Growth Regulators

References

  • Ahire AE, Laddha KS (2002) Beta amyrin palmitate- isolation from Couroupita guianensis aubl. Leaves. Indian Drugs 39:216–216

    Google Scholar 

  • Arikat NA, Jawad FM, Karam NS, Shibli RA (2004) Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa mill.). Sci Hortic 100:193–202

    CAS  Article  Google Scholar 

  • Arora K, Sharma M, Srivastava J, Ranade SA, Sharma AK (2010) Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica a. Juss. (neem) employing nodal stem segments. Agrofor Syst 78:53–63

    Article  Google Scholar 

  • Arya V, Singh RP, Shekhawat NS (2002) A micropropagation protocol for mass multiplication and off-site conservation of Celastrus paniculatus- a vulnerable medicinal plant of India. J Sustain For 14:107–120

    Article  Google Scholar 

  • Baskaran P, Van Staden J (2013) Rapid in vitro micropropagation of Agapanthus praecox. S Afr J Bot 86:46–50

    CAS  Article  Google Scholar 

  • Chirinea CF, Pasqual M, Araujo AG de, Pereira AR, Castro EM de (2012) Acclimatization and leaf anatomy of micropropagated Fig plants. Rev Bras Frutic Jaboticabal – SP 34:1180–1188

  • Compton ME, Mize CW (1999) Statistical considerations for in vitro research: I -birth of an idea to collecting data. In Vitro Cell Dev Biol Plant 35:115–121

  • Deepa HR (2007) Puducherry comes out with list of state symbols. The Hindu, Sat. 21, Chennnai, TN, India

  • Desal T, Golatakar SG, Rane JB, Ambaye RY, Kamath VR (2003) Larvicidal property of Couroupita guianensis aubl. Indian Drugs 40:484–486

    Google Scholar 

  • Dhavala A, Rathore TS (2010) Micropropagation of Embelia ribes burm F. through proliferation of adult plant axillary shoots. In Vitro Cell Dev Biol Plant 46:180–191

  • Gousia SK, Ashok KK, Vinay KT, Naveena LL (2013) Biological activities and medicinal properties of Couroupita guianensis. Int J Pharm Pharmaceut Sci Res 3(4):140–143

    Google Scholar 

  • Jayashree BR, Severina JV, Suprabha GG, Ambaye RY, Khadse BG (2001) Chemical examination of the flowers of Couroupita guianensis aubl. Indian J Pharm Sci 63:72–73

    Google Scholar 

  • Jiang Q, Zhang Y, Zhong C, Zeng B, Bogusz D, Franche C (2012) Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. Via indirect organogenesis. New For 43:143–154

    Article  Google Scholar 

  • Johansen DA (1940) Plant microtechnique. McGraw Hill Co, New York

    Google Scholar 

  • John M, Sanda SI, Amasino AM (1997) Analysis of flowering time in ecotypes of Arabidopsis thaliana. J Hered 88:69–72

    Article  PubMed  Google Scholar 

  • Kavitha R, Kamalakannan P, Deepa T, Elamathi R, Sridhar S, Suresh KJ (2011) In vitro antimicrobial activity and phytochemical analysis of Indian medicinal plant Couroupita guianensis aubl. J Chem Pharm Res 3:115–121

    Google Scholar 

  • Kevers C, Franck T, Strasser RJ, Dommes J, Gaspar T (2004) Hyperhydricity of micropropagated shoots; a typically stress-induced change of physiological state. Plant Cell Tissue Organ Cult 77:181–191

    Article  Google Scholar 

  • Khan MR, Kihara M, Omoloso AD (2003) Antibiotic activity of Couroupita guianensis. J Herbs Spices Med Plant 10:95–108

    Article  Google Scholar 

  • Lim TK (2012) Couroupita guianensis. Edible medicinal and nonmedicinal plants fruits, Springer Verlag, Berlin Heidelberg

    Book  Google Scholar 

  • Lloyd G, McCown BG (1980) Commercially feasible micropropagation of mountain laurel Kalmia latifolia by use of shoot -tip culture. Int Plant Propagator’s Soc Combin Proc 30:421–427

    Google Scholar 

  • Lodha D, Patel A, Shekhawat NS (2015) A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub. Physiol Mol Biol Plants. doi:10.1007/s12298-015-0310-6

    PubMed  Google Scholar 

  • Mariappan P, Srinivasan R, Kandasamy K (2012) Antioxidant and anticancer activities of isatin (1 H-indole-2,3-dione), isolated from the flowers of Couroupita guianensis aubl. Indian J Med Res 136:822–826

    Google Scholar 

  • Misic D, Grubisic D, Konjevic R (2006) Micropropagation of salvia brachyodon through nodal explants. Biol Plant 50:473–476

    Article  Google Scholar 

  • Mitre M 2012 Couroupita guianensis. In: IUCN Red List of Threatened Species. Version 2012.2. http://www.iucnredlist.org

  • Muniswamy B, Sreenath HL (2000) In-Vitro development of plants from cultured embryos of Cannon Ball Tree (Couroupita guianensis Aubl) Indian. J For 23:202–204

    Google Scholar 

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

    CAS  Article  Google Scholar 

  • Pospisilova J, Synkova H, Rulcova J (2000) Cytokinins and water stress. Biol Plant 43:321–328

  • Panwar D, Ram K, Harish SNS (2012) In vitro propagation of Eulophia nuda lindl., an endangered orchid. Sci Hortic 139:46–52

    CAS  Article  Google Scholar 

  • Patel AK, Phulwaria M, Rai MK, Gupta AK, Shekhawat S, Shekhawat NS (2014) In vitro propagation and ex vitro rooting of Caralluma edulis (edgew.) benth. & hook. f. An endemic and endangered edible plant species of the Thar desert. Sci Hortic 165:175–180

    CAS  Article  Google Scholar 

  • Phulwaria M, Rai MK, Patel AK, Kataria V, Shekhawat NS (2013) A genetically stable Rooting protocol for propagating a threatened medicinal plant-Celastrus paniculatus. Appl Biochem Biotechnol 170:1163–1173

    CAS  Article  PubMed  Google Scholar 

  • Pradhan D, Panda PK, Tripathy G (2009) Evaluation of the immune modulatory activity of the methanolic extract of Couroupita guianensis aubl. Flowers in rats. Nat Prod Radiance 8:37–42

    Google Scholar 

  • Rai Y (2014) Early seedling growth status of threatened medicinal tree species Couroupita guianensis aubl. In district Meerut, (U.P.) India. Int J Inn Sci Res 8:252–255

    Google Scholar 

  • Rajamanickam V, Rajasekaran A, Darlin quine S, Jesupillai M, Sabitha R (2009) Anthelmintic activity of the flower extract of Couroupita guianensis. Int J Alt Med 8:107–111

    Google Scholar 

  • Rane JB, Vahanwala SJ, Golatkar SG, Ambaye RY, Khadse BG (2001) Chemical examination of the flowers of Couroupita guianensis aubl. Indian J Pharm Sci 63:72–73

    Google Scholar 

  • Ranaweeraa KK, Gunasekaran MTK, Eeswara JP (2013) Ex vitro rooting: A low cost micropropagation technique for Tea (Camellia sinensis (L.) O. Kuntz.) hybrids. Sci Hortic 155:8–14

  • Rathore NS, Rathore N, Shekhawat NS (2013) In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana. Acta Physiol Plant. doi:10.1007/s11738-013-1301-2

    Google Scholar 

  • Regina V (2014) Pharmacognostical studies of various parts of Couroupita guianensis aubl. Int J Curr Res Biosci Plant Biol 1:17–26

    Google Scholar 

  • Richter HG, Dallwitz MJ (2000) Onwards Commercial timbers: descriptions, illustrations, identification, and information retrieval In English, French, German, Portuguese, and Spanish Version: 25th June 2009. http://delta-intkey.com

  • Sai KC, Gaddala N, Vanamala S, Naresh V, Elumalai A (2011) A short review on therapeutic uses of Couroupita guianensis aubl. Int Res J Pharm Appl Sci 1:105–108

    Google Scholar 

  • Santosh KS (2011) Cauliflory and cannon ball tree. Sci Report 48:53–55

    Google Scholar 

  • Santosh AS, Gourav NS, Kavita DP, Vaibhavi SP, Samir SW, Suresh GK (2013) Estimation of tannins present in fruit pulp and fruit shell of Couroupita guianensis. J Pharm Res 2:18–20

    Google Scholar 

  • Sass JE (1940) Elements of botanical microtechnique. McFraw-Hill Book Co, New York and London

    Google Scholar 

  • Sathe SB, Jadhav AP, Sonawane AA, More SH, Kadam V (2013) Pharmacognostical evaluation of Couroupita guianensis aubl. Leaf. Indo American J Pharm Res 3:6119–6124

    Google Scholar 

  • Shah GN, Shete SA, Patil VS, Patil KD, Killedar SG (2012) Standardization and anti-bacterial activity of Couroupita guianensis fruit pulp extract. Int J Pharmacog Phytochem Res 4:85–89

    Google Scholar 

  • Shekhawat MS, Kannan N, Manokari M, Ramanujam MP (2014) En efficient micropropagation protocol for high-frequency plantlet regeneration from liquid culture of nodal tissues in a medicinal plant Turnera ulmifolia L. J Sustain For 33:327–336

    Article  Google Scholar 

  • Shekhawat MS, Kannan N, Manokari M (2015a) In vitro propagation of traditional medicinal and dye yielding plant Morinda coreia Buch. -Ham. S Afr J Bot. doi:10.1016/j.sajb.2015.05.018

    Google Scholar 

  • Shekhawat MS, Kannan N, Manokari M, Ravindran CP (2015b) Enhanced micropropagation protocol of Morinda citrifolia L. through nodal explants. J Appl Res Med Aromat Plant. doi:10.1016/j.jarmap.2015.06.002

    Google Scholar 

  • Shete SA, Shah GN, Walke SS, Patil VS, Patil KD, Killedar SG (2013) Standardization and anti bacterial activity of Couroupita guianensis fruit shell extract. Int J Bio 2:360–364

    Google Scholar 

  • Singh J, Tiwari KN (2012) In vitro plant regeneration from decapitated embryonic axes of Clitoria ternatea L.-an important medicinal plant. Ind Crop Prod 35:224–229

    CAS  Article  Google Scholar 

  • Sivakumar T, Shankar T, Vijayabaskar P, Geetha G (2012) Efficacy of Couroupita guianensis against selected human pathogens. Adv Biol Res 6:59–63

    CAS  Google Scholar 

  • Smith RH (2000) Plant tissue culture: techniques and experiments. Academic Press, Tokyo

    Google Scholar 

  • Swamy RBV, Himabindu K, Lakshmi sita G (2002) In Vitro micropropagatipon of Elite Rosewood (Dalbergia latifolia Roxb). Plant Cell Rep 11:126–131

    Google Scholar 

  • Thomas TD (2008) The role of activated charcoal in plant tissue culture. Biotechnol Adv 26:618–631

    CAS  Article  PubMed  Google Scholar 

  • Umachigi SP, Jayaveera KN, Ashok KCK, Kumar GS (2007) Antimicrobial, wound healing, and antioxidant potential of Couroupita guianensis in rats. Pharmacol Online 3:269–281

    Google Scholar 

  • Velliangiri P, Subban R (2012) Quantification of quercetin and stigmasterol of Couroupita guianensis aubl by HPTLC method and in-vitro cytototoxic activity by mtt assay of the methanol extract against Hela, nih 3 t3 and hepg2 cancer cell lines. Int J Pharm Pharmac Sci 4:126–130

    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

    CAS  Article  Google Scholar 

  • Yokota S, KArim MZ, Azad MAK, Rahman MM, Eizawa J, Saito Y, Yshiguri F, Iizuka K, Yahara S, Yoshizwan (2007) Histological observation of changes in leaf structure during successive micropropagation stages in Aralia elata and Phellodendron amurense. Plant Biol 24:221–226

    Google Scholar 

Download references

Acknowledgments

Authors are grateful to the Department of Science, Technology and Environment, Government of Puducherry, India for providing financial support under Grant-In-Aid Scheme [No. 10/DSTE/GIA/RP/JSA-I/2013/213] for Couroupita guianensis Aubl.

Author information

Authors and Affiliations

  1. Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C., Mahe, Mahe, Pondicherry, India

    Mahipal S. Shekhawat & M. Manokari

Authors
  1. Mahipal S. Shekhawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Manokari
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mahipal S. Shekhawat.

Ethics declarations

Financial disclosures

MSS has received financial support under Grant-In-Aid Scheme of The DST&E, Government of Puducherry, India.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shekhawat, M.S., Manokari, M. In vitro propagation, micromorphological studies and ex vitro rooting of cannon ball tree (Couroupita guianensis aubl.): a multipurpose threatened species. Physiol Mol Biol Plants 22, 131–142 (2016). https://doi.org/10.1007/s12298-015-0335-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-015-0335-x

Keywords

  • Couroupita guianensis
  • Micropropagation
  • Ex vitro rooting
  • Acclimatization
  • Micromorphology
  • Conservation