Effect of colchicine induced tetraploidy on morphology, cytology, essential oil composition, gene expression and antioxidant activity of Citrus limon (L.) Osbeck

  • Gunasekaran Bhuvaneswari
  • Ramaraj ThirugnanasampandanEmail author
  • Madhusudhanan Gogulramnath
Research Article


Citrus limon (L.) Osbeck cultivated all over the world is a valuable source of aromatic essential oil. To develop tetraploids of C. limon, four different concentration of colchicine (0.025, 0.05, 0.1 and 0.2%; w/v) and three varied exposure time (12, 24 and 48 h) were employed. The ploidy level of diploids (2n = 2x = 18) and tetraploids (2n = 4x = 36) were determined by direct chromosome count and confirmed by flow cytometric analyses. Successful result with maximum tetraploidy frequency was observed in plantlets developed from seeds treated with 0.025% colchicine for 24 h. Morphological and stomatal characteristics indicated that tetraploids were taller with increased leaf and root length. On the other side, the leaves of tetraploids had fewer and larger stomata with a greater number of chloroplasts in guard cells in contrast with diploids. GC–GC/MS analyses showed cyclic monoterpene, limonene had increased significantly in tetraploids and was further confirmed by HPLC quantification. RT-PCR analyses revealed unaltered expression of monoterpene synthase, sesquiterpene synthase and flavone synthase and remarkable upregulation of genes such as limonene synthase, chalcone synthase and phenylalanine ammonia lyase in tetraploids. Antioxidant activity of essential oil of tetraploids was higher than diploids in all the five test systems studied. Overall, the findings of the present study prove that colchicine induced tetraploidy in C. limon could be a greater source of essential oil with improved composition and of economic significance.


Lemon Colchicine Flow cytometry Terpenes Limonene GC/MS HPLC RT-PCR 



Murashige and Skoog


Gas chromatography


Gas chromatography/mass spectrometry


High performance liquid chromatography


Reverse transcription-polymerase chain reaction


Monoterpene synthase


Limonene synthase


Sesquiterpene synthase


Flavone synthase


Chalcone synthase


Phenylalanine ammonia lyase





The authors wish to sincerely acknowledge SIF-VIT University (Vellore), CSIF-IIISM SRM Institute of Science and Technology (Katankulathur) and SAIF ICAR-SBI (Coimbatore) for providing instrumentation facilities.

Author contributions

GB: collection of plant material, performance of experiments, data collection and manuscript preparation. RT: experimental outline, data interpretation. MG: experimental design development.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12298_2019_718_MOESM1_ESM.doc (46 kb)
Supplementary material 1 (DOC 45 kb)


  1. Abdoli M, Moieni A, Badi HN (2013) Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.). Acta Physiol Plant 35(7):2075–2083CrossRefGoogle Scholar
  2. Aruoma OI (1994) Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol 32:671–683CrossRefGoogle Scholar
  3. Ben Hsouna A, Ben Halima N, Smaoui S, Hamdi N (2017) Citrus limon essential oil: chemical composition, antioxidant and antimicrobial activities with its preservative effect against Listeria monocytogenes inoculated in minced beef meat. Lipids Health Dis 16:146CrossRefGoogle Scholar
  4. Bosco SFD, Tusa N, Conicella C (1999) Microsporogenesis in a Citrus interspecific tetraploid somatic hybrid and its fusion parents. Heredity 83:373–377CrossRefGoogle Scholar
  5. Cataldo F, Keheyan Y, Baccaro S (2004) Gamma-radiolysis of chiral molecules: R(+)-limonene, S(−)-limonene and R(−)-α-phellandrene. J Radioanal Nucl Chem 262(2):423–428CrossRefGoogle Scholar
  6. Chew YL, Goh JK, Lim YY (2009) Assessment of in vitro antioxidant capacity and polyphenolic composition of selected medicinal herbs from Leguminosae family in Peninsular Malaysia. Food Chem 116:13–18CrossRefGoogle Scholar
  7. Choi B, Kang S, Ha Y, Park G, Ackman RG (2002) Conjugated linoleic acid as a supplemental nutrient for common carp (Cyprinus carpio). Food Sci Biotechnol 11:457–461Google Scholar
  8. Dhawan OP, Lavania UC (1996) Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica 87(2):81–89CrossRefGoogle Scholar
  9. Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tissue Organ Cult 104:359–373CrossRefGoogle Scholar
  10. Ewald D, Ulrich K, Naujoks G, Schroder MB (2009) Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro. Plant Cell Tissue Organ Cult 99:353–357CrossRefGoogle Scholar
  11. FAO (2017) FAOSTAT. Accessed 20 May 2019
  12. Gantait S, Mandal N, Bhattacharyya S, Das PK (2011) Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella. Plant Cell Tissue Organ Cult 106:485–493CrossRefGoogle Scholar
  13. Goswami K, Sharma R, Singh PK, Singh G (2013) Micropropagation of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) and assessment of genetic fidelity of micropropagated plants using RAPD markers. Physiol Mol Biol Plants 19(1):137–145CrossRefGoogle Scholar
  14. Hannweg K, Visser G, de Jager K, Bertling I (2016) In vitro-induced polyploidy and its effect on horticultural characteristics, essential oil composition and bioactivity of Tetradenia riparia. S Afr J Bot 106:186–191CrossRefGoogle Scholar
  15. Lavania UC (2005) Genomic and ploidy manipulation for enhanced production of phyto-pharmaceuticals. Plant Genet Resour 3(2):170–177CrossRefGoogle Scholar
  16. Lee S, Umano K, Shibamoto T, Lee K (2005) Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem 91:131–137CrossRefGoogle Scholar
  17. Levy M (1976) Altered glycoflavone expression in induced autotetraploids of Phlox dummondii. Biochem Syst Ecol 4:249–254CrossRefGoogle Scholar
  18. Lin X, Zhou Y, Zhang J, Lu X, Zhang F, Shen Q, Wu S, Chen Y, Wang T, Tang K (2011) Enhancement of artemisinin content in tetraploid Artemisia annua plants by modulating the expression of genes in artemisinin biosynthetic pathway. Biotechnol Appl Biochem 58(1):50–57CrossRefGoogle Scholar
  19. Mukhopadhyay D, Dasgupta P, Roy DS, Palchoudhuri S, Chatterjee I, Ali S, Dastidar SG (2016) A sensitive in vitro spectrophotometric hydrogen peroxide scavenging assay using 1,10-phenanthroline. Free Radic Antioxid 6:124–132CrossRefGoogle Scholar
  20. Murashige T, Skoog FM (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  21. Mustafa NE (2015) Citrus essential oils: current and prospective uses in the food industry. Recent Pat Food Nutr Agric 7(2):115–127CrossRefGoogle Scholar
  22. NIST/NIH/EPA (2008) Mass spectral library with search program data version: NIST v08. Standard reference data program, National Institute of Standards and Technology, GaithersburgGoogle Scholar
  23. Noori SAS, Norouzi M, Karimzadeh G, Shirkool K, Niazian M (2017) Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan (Trachyspermum ammi L.). Plant Cell Tissue Organ Cult 130(3):543–551CrossRefGoogle Scholar
  24. Orbovic V, Calovic M, Viloria Z, Nielsen B, Gmitter FG Jr, Castle WS, Grosser JW (2008) Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry. Euphytica 161:329–335CrossRefGoogle Scholar
  25. Perez-Tornero O, Tallon CI, Porras I (2010) An efficient protocol for micropropagation of lemon (Citrus limon) from mature nodal segments. Plant Cell Tissue Organ Cult 100:263–271CrossRefGoogle Scholar
  26. Pessina D, Gentili R, Barcaccia G, Nicole S, Rossi G, Barbesti S, Sgorbati S (2011) DNA content, morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus. Sci Hortic 129:663–673CrossRefGoogle Scholar
  27. Planchais S, Glab N, Inze D, Bergonioux C (2000) Chemical inhibitors: a tool for plant cell cycle studies. FEBS Lett 476:78–83CrossRefGoogle Scholar
  28. Ramsey J, Schemske DW (1998) Pathways, mechanisms, and rates of polyploidy formation in flowering plants. Annu Rev Ecol Syst 29:467–501CrossRefGoogle Scholar
  29. Romero-Aranda R, Bondada BR, Syvertsen JP, Grosser JW (1997) Leaf characteristics and net gas exchange of diploid and autotetraploid Citrus. Ann Bot 79:153–160CrossRefGoogle Scholar
  30. Sattler MC, Carvalho CR, Clarindo WR (2016) The polyploidy and its key role in plant breeding. Planta 243(2):281–296CrossRefGoogle Scholar
  31. Siddique AB, Mizanur Rahman SM, Hossain MA (2016) Chemical composition of essential oil by different extraction methods and fatty acid analysis of the leaves of Stevia rebaudiana bertoni. Arab J Chem 9:S1185–S1189CrossRefGoogle Scholar
  32. Soltis DE, Albert VA, Leebens-Mack J, Bell CD, Paterson AH, Zheng C, Sankoff D, de Pamphilis CW, Wall PK, Soltis PS (2009) Polyploidy and angiosperm diversification. Am J Bot 96:336–348CrossRefGoogle Scholar
  33. Song C, Liu S, Xiao J, He W, Zhou Y, Qin Q, Zhang C, Liu Y (2012) Polyploid organisms. Sci China Life Sci 55:301–311CrossRefGoogle Scholar
  34. Tang ZQ, Chen DL, Song ZJ, He YC, Cai DT (2010) In vitro induction and identification of tetraploid plants of Paulownia tomentosa. Plant Cell Tissue Organ Cult 102:213–220CrossRefGoogle Scholar
  35. Tauchen J, Doskocil I, Caffi C, Lulekal E, Marsik P, Havlik J, Damme PV, Kokosk L (2015) In vitro antioxidant and anti-proliferative activity of Ethiopian medicinal plant extracts. Ind Crops Prod 74:671–679CrossRefGoogle Scholar
  36. Tavan M, Mirjalili MH, Karimzadeh G (2015) In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae). Plant Cell Tissue Organ Cult 122(3):573–583CrossRefGoogle Scholar
  37. Turek C, Stintzing FC (2013) Stability of essential oils: a review. Compr Rev Food Sci Food Saf 12:40–53CrossRefGoogle Scholar
  38. Xu C, Huang Z, Liao T, Li Y, Kang X (2016) In vitro tetraploid plants regeneration from leaf explants of multiple genotypes in Populus. Plant Cell Tissue Organ Cult 125(1):1–9CrossRefGoogle Scholar
  39. Zheljazkov VD, Astatkie T, Jeliazkova EA, Schlegel V (2012) Distillation time alters essential oil yield, composition, and antioxidant activity of male Juniperus scopulorum trees. J Oleo Sci 61:641–648CrossRefGoogle Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  • Gunasekaran Bhuvaneswari
    • 1
  • Ramaraj Thirugnanasampandan
    • 2
    Email author
  • Madhusudhanan Gogulramnath
    • 1
  1. 1.PG and Research Department of BiotechnologyKongunadu Arts and Science CollegeCoimbatoreIndia
  2. 2.PG and Research Department of BotanyKongunadu Arts and Science CollegeCoimbatoreIndia

Personalised recommendations