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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
  • 22 Downloads

Abstract

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.

Keywords

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

Abbreviations

MS

Murashige and Skoog

GC

Gas chromatography

GC/MS

Gas chromatography/mass spectrometry

HPLC

High performance liquid chromatography

RT-PCR

Reverse transcription-polymerase chain reaction

MTS

Monoterpene synthase

LS

Limonene synthase

STS

Sesquiterpene synthase

FS

Flavone synthase

CHS

Chalcone synthase

PAL

Phenylalanine ammonia lyase

DPPH

4′,6-Diamidino-2-phenylindole

Notes

Acknowledgements

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)

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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

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