Skip to main content

Advertisement

SpringerLink
Log in

Micropropagation and molecular characterization of Thymus sibthorpii Benth. (Lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern

  • Micropropagation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

Thymus sibthorpii Benth. (Lamiaceae), with accession number 01,1796-22, is a biotype of native Greek thyme with ascending stems and potential use as a new medicinal-aromatic crop and ornamental plant. An efficient and reliable protocol for in vitro clonal propagation of T. sibthorpii from nodes and meristem tip explants was developed. Shoot proliferation succeeded on a new basal medium (BB) without plant growth regulators, as prior experiments with 6-benzyladenine generated hyperhydricity. Eight different basal media were compared; on two formulations using the new BB 5.9 and 5.6 shoots per explant were produced. Regenerated single shoots were rooted in the BB medium, supplemented with 5 μM of indole-3-butyric acid, and produced 3.1 roots along with 2.5 adventitious shoots. Three types of acclimatization were assessed: in vitro, using two different systems (no significant differences); ex vitro, using eight soil substrates under greenhouse and outdoor nursery conditions (in two of them, 100% of plantlets survived); and in field cultivations, established at eight geographically distant areas of Greece (100% survival rate at all locations). Molecular characterization of T. sibthorpii was evaluated with one nuclear ribosomal DNA and seven chloroplast DNA markers, followed by DNA sequence comparisons with a total of 30 different Thymus species, subspecies, and varieties. The trnH/psbA, trnL/trnF, and matK genes were the most efficient markers for molecular characterization of T. sibthorpii. The molecular markers rpoC1 and petB/petD did not match to any Thymus species and therefore, these DNA sequences provide new sequence information for entire Thymus taxa.

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

Figure 1.
Figure 2.

Similar content being viewed by others

References

  • Abraham EM, Aftzalanidou A, Ganopoulos I, Osathanunkul M, Xanthopoulou A, Avramidou E, Sarrou E, Aravanopoulos F, Madesis P (2018) Genetic diversity of Thymus sibthorpii Bentham in mountainous natural grasslands of Northern Greece as related to local factors and plant community structure. Ind Crops Prod 111:651–659

    Article  Google Scholar 

  • Bakhtiar Z, Mirjalili MH, Sonboli A (2016) In vitro callus induction and micropropagation of Thymus persicus (Lamiaceae), an endangered medicinal plant. Crop Breed Appl Biotechnol 16:48–54

    Article  CAS  Google Scholar 

  • Bakhtiar Z, Mirjalili MH, Sonboli A, Farimani MM, Ayyari M (2014) In vitro propagation, genetic and phytochemical assessment of Thymus persicus – a medicinally important source of pentacyclic triterpenoids. Biologia 69:594–603

  • Beheshti HB, Morteza K (2005) Effects of media and growth regulators on garden thyme (Thymus vulgaris L.) micropropagation. Iran J Hortic Sci Technol 6:61–68

    Google Scholar 

  • Bernard F, Moghadam N, Mirzajani F (2015) The effect of colloidal silver nanoparticles on the level of lignification and hyperhydricity syndrome in Thymus daenensis vitro shoots: a possible involvement of bonded polyamines. In Vitro Cell Dev Biol–Plant 51:546–553

    Article  CAS  Google Scholar 

  • Bertoli A, Sárosi S, Bernáth J, Pistelli L (2010) Characterization of some Italian ornamental Thyme by their aroma. Nat Prod Commun 5:291–296

    CAS  PubMed  Google Scholar 

  • Casiraghi M, Labra M, Ferri E, Galimberti A, De Mattia F (2010) DNA barcoding: a six-question tour to improve users’ awareness about the method. Brief Bioinform 11:440–453

    Article  CAS  PubMed  Google Scholar 

  • Chase W, Cowan MR, Hollingsworth M, van den Berg PC, Madriñán S, Petersen G, Seberg O, Jorgsensen T, Cameron K, Carine M, Pedersen N, Hedderson T, Conrad F, Salazar AG, Richardson JE, Hart M, Barraclough T, Laura K, Wilkinson M (2007) A proposal for a standardized protocol to barcode all land plants. Taxon 56:295–299

    Article  Google Scholar 

  • Coelho N, Gonçalves S, Elena M, Benito G, Romano A (2012) Establishment of an in vitro propagation protocol for Thymus lotocephalus, a rare aromatic species of the Algarve (Portugal). Plant Growth Regul 66:69–74

    Article  CAS  Google Scholar 

  • De Mattia F, Bruni I, Galimberti A, Cattaneo F, Casiraghi M, Labra M (2011) A comparative study of different DNA barcoding markers for the identification of some members of Lamiaceae. Food Res Int 44:693–702

    Article  CAS  Google Scholar 

  • De Mattia F, Gentili R, Bruni I, Galimberti A, Sgorbati S, Casiraghi M, Labra M (2012) A multi-marker DNA barcoding approach to save time and resources in vegetation surveys. Bot J Linn Soc 169:518–529

    Article  Google Scholar 

  • Delcheh SK, Kashefi B, Mohammadhassan R (2014) A review optimization of tissue culture medium medicinal plant: thyme. Int J Farm Alli Sci 3:1015–1019

    Google Scholar 

  • Dimopoulos P, Raus T, Bergmeier E, Constantinidis Th, Iatrou G, Kokkini S, Strid A, Tzanoudakis D (2013) Vascular plants of Greece: an annotated checklist. Englera 31. Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freie Universität Berlin / Hellenic Botanical Society, Athens, Greece

  • Echeverrigaray S, Agostini G, Ati-Serini L, Paroul N, Pauleti GF, Santos AC (2001) Correlation between the chemical and genetic relationships among commercial thyme cultivars. J Agric Food Chem 49:4220–4223

    Article  CAS  PubMed  Google Scholar 

  • El-Makawy M, Yasser M, Abdallah M, Nishawy S (2008) In vitro clonal propagation of Thymus capitatus L. through direct regeneration. Pak J Biotechnol 5:39–44

    Google Scholar 

  • Emami SD, Hosseini NM (2008) Cultivation and production of certain herbs and spices, 2nd edn. University of Tehran Press, Tehran

    Google Scholar 

  • Fazekas AJ, Burgess KS, Kesanakurti PR, Graham SW, Newmaster SG, Husband BC, Percy DM, Hajibabaei M, Barrett SC (2008) Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS One 3:e2802

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Federici S, Galimberti A, Bartolucci F, Bruni I, de Mattia F, Cortis P, Labra M (2013) DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae). Bot J Linn Soc 171:687–699

    Article  Google Scholar 

  • Fraternale D, Giamperi L, Ricci D, Rocchi MBL, Guidi L, Epifano F, Marcotullio MC (2003) The effect of triacontanol on micropropagation and on secretory system of Thymus mastichina. Plant Cell Tissue Organ Cult 74:87–97

    Article  CAS  Google Scholar 

  • Furmanowa M, Olszowska O (1980) Thymus vulgaris L. propagation through tissue culture. Acta Pol Pharm 37:243–247

    Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Günaydin M, Laghari HA, Ersan B, Münevver S, Atalay S (2017) Accumulation of phenolics in natural and micropropagated plantlets of Thymus pseudopulegioides Klokov & Des.-Shost. with their antioxidant potentials. Turk J Biol 41:754–764

    Article  CAS  Google Scholar 

  • Hadian J, Bigdeloo M, Nazeri V, Khadivi-Khub A (2014) Assessment of genetic and chemical variability in Thymus caramanicus. Mol Biol Rep 41:3201–3210

    Article  CAS  PubMed  Google Scholar 

  • Hammer N, Loffler S, Feja C, Sandrock M, Schmidt W, Bechmann I, Steinke H (2012) Ethanol-glycerin fixation with thymol conservation: a potential alternative to formaldehyde and phenol embalming. Anat Sci Educ 5:225–233

    Article  PubMed  Google Scholar 

  • Hassannejad S, Bernard F, Mirzajani F, Gholami M (2012) SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changes. Plant Physiol Biochem 51:40–46

    Article  CAS  PubMed  Google Scholar 

  • Hollingsworth ML, Clark A, Forrest LL, Richardson JE, Pennington RT, Long DG, Cowan R, Chase MW, Gaudeul M, Hollingsworth PM (2009) Selecting barcoding loci for plants: evaluation of seven candidate loci with species level sampling in three divergent groups of land plants. Mol Ecol Resour 9:439–457

    Article  CAS  PubMed  Google Scholar 

  • Hopkins WG (1999) Introduction to plant physiology, 4th edn. Wiley, Hoboken ISBN: 978-0470247662

    Google Scholar 

  • Hosseini BB, Khoshkhoy M (2005) Effects of media and growth regulators on garden thyme (Thymus vulgaris L.) micropropagation. Iran J Hortic Sci Technol 6:61–68

    Google Scholar 

  • Hosseinzadeh S, Jafarikukhdan A, Hosseini A, Armand R (2015) The application of medicinal plants in traditional and modern medicine: a review of Thymus vulgaris. International J Clin Med 6:635–642

    Article  Google Scholar 

  • Karaca M, Ince AG, Aydin A, Elmasulu SY, Turgut K (2015) Microsatellites for genetic and taxonomic research on thyme (Thymus L.). Turk J Biol 39:147–159

    Article  CAS  Google Scholar 

  • Karalija E, Parić A (2011) The effect of BA and IBA on the secondary metabolite production by shoot culture of Thymus vulgaris L. Biol Nyssana 2:29–35

    Google Scholar 

  • Kaya E (2010) Development of efficient micropropagation and cryopreservation protocols for Thymus germplasm. In vitro biology meeting held in collaboration with the 12th IAPB congress, P283

  • Khoshsokhan F, Babalar M, Fatahi M, Poormeidani A (2014) Assessment of genetic diversity of some wild populations of Thymus kotschyanus using RAPD molecular markers. Cercetări Agronom Moldova 3:71–81

    Article  Google Scholar 

  • Kress JW, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcl gene complements the non-coding trnH-psbA spacer region. PLoS One 2(6):e508

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005) Use of DNA barcodes to identify flowering plants. Proc Natl Acad Sci U S A 102:8369–8374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krigas N, Maloupa E (2008) The Balkan Botanic Garden of Kroussia, Northern Greece: a garden dedicated to the conservation of native plants of Greece and the Balkans. Sibbaldia 6:9–27

    Google Scholar 

  • Kulpa D, Wesołowska A, Jadczak P (2018) Micropropagation and composition of essentials oils in garden thyme (Thymus vulgaris L.). Not Bot Horti Agrobo 46:525–532

    Article  CAS  Google Scholar 

  • Leal F, Taghouti M, Nunes F, Amélia S, Coelho AC, Matos M (2017) Thymus plants: a review—micropropagation, molecular and antifungal activity. In: El-Shemy HA (ed) Active ingredients from aromatic and medicinal plants. IntechOpen, London, U.K, pp 107–126

    Google Scholar 

  • Ledford H (2008) Botanical identities: DNA barcoding for plants comes a step closer. Nature 451:616

    Article  CAS  PubMed  Google Scholar 

  • Lee J, Baldwin BG, Gottlieb LD (2002) Phylogeny of Stephanomeria and related genera (Compositae-Lactuceae) based on analysis of 18S-26S nuclear rDNA ITS and ETS sequences. Am J Bot 89:160–168

    Article  CAS  PubMed  Google Scholar 

  • Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127

    Article  CAS  Google Scholar 

  • Löhne C, Borsch T (2005) Phylogenetic utility and molecular evolution of the petD group II intron in basal angiosperms. Mol Biol Evol 22:317–332

    Article  PubMed  CAS  Google Scholar 

  • Maloupa E, Krigas N, Grigoriadou K, Lazari D, Tsoktouridis G (2008) Conservation strategies for native plant species and their sustainable exploitation: the case of the Balkan Botanic Garden of Kroussia, N. Greece. In: Teixeira da Silva JA (ed) Floriculture ornamental plant biotechnology: advances and topical issues (1st edition), Vol. V (4), Global Science Books, pp 37–56

  • Marco-Medina A, Casas JL (2015) In vitro multiplication and essential oil composition of Thymus moroderi Pau ex Martinez, an endemic Spanish plant. Plant Cell Tissue Organ Cult 120:99–108

    Article  CAS  Google Scholar 

  • Mendes MD, Figueiredo AC, Oliveira MM, Trindade H (2013) Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius. Plant Cell Tissue Organ Cult 113:341–351

    Article  CAS  Google Scholar 

  • Mendes ML, Romano A (1999) In vitro cloning of Thymus mastichina L. field-grown plants. Acta Hortic 502:303–306

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

    Article  CAS  Google Scholar 

  • Nabavi SM, Marchese A, Izadi M, Curti V, Daglia M, Nabavi SF (2015) Plants belonging to the genus Thymus as antibacterial agents: from farm to pharmacy. Food Chem 173:339–347

    Article  CAS  PubMed  Google Scholar 

  • Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 169:85–87

    Article  Google Scholar 

  • Nordine A, Bousta D, El Khanchoufi A, El Meskaoui A (2013a) An efficient and rapid in vitro propagation system of Thymus hyemalis Lange, a wild medicinal and aromatic plant of Mediterranean region. Int J Pharma Biosci Technol 1:118–129

  • Nordine A, El Meskaoui A (2014) Rapid in vitro regeneration and clonal multiplication of Thymus bleicherianus Pomel, a rare and threatened medicinal and aromatic plant in Morocco. Med Aromat Plants 3:145. https://doi.org/10.4172/2167-0412.1000145

  • Nordine A, Rachida TC, El Meskaoui A (2013b) Micropropagation of Thymus satureioides Coss. an endangered medicinal plant of Morocco. J Agric Sci Technol 9:487–501

    Google Scholar 

  • Ozudogru EA, Kaya E, Kirdok E, Issever-Ozturk S (2011) In vitro propagation from young and mature explants of thyme (Thymus vulgaris and T. longicaulis) resulting in genetically stable shoots. In Vitro Cell Dev Biol–Plant 47:309–320

    Article  Google Scholar 

  • Pérez-Tortosa V, López-Orenes A, Martínez-Pérez A, Ferrer MA, Calderón AA (2012) Antioxidant activity and rosmarinic acid changes in salicylic acid-treated Thymus membranaceus shoots. Food Chem 130:362–369

    Article  CAS  Google Scholar 

  • Pierik RLM (1999) In Vitro culture of higher plants, 4th edn. Springer, Netherlands ISBN: 978-0792352679

    Google Scholar 

  • Pluhár Z, Kocsis M, Kuczmog A, Csete S, Simkó H, Sárosi S, Molnár P, Horváth G (2012) Essential oil composition and preliminary molecular study of four Hungarian Thymus species. Acta Biol Hung 63:81–96

    Article  PubMed  CAS  Google Scholar 

  • Rustaiee AR, Yavari A, Nazeri V, Shokrpour M, Seidkon F, Rasouli M (2013) Genetic diversity and chemical polymorphism of some Thymus species. Chem Biodivers 10:1088–1098

    Article  CAS  PubMed  Google Scholar 

  • Sáez F, Sknchez P, Piqueras A (1994) Micropropagation of Thymus piperella. Plant Cell Tissue Organ Cult 39:269–272

    Article  Google Scholar 

  • Sargsyan E, Vardanyan A, Ghalachyan L, Bulgadaryan S (2011) Cultivation of Thymus by in vitro and hydroponics combined method. World Acad Sci Eng Technol 80:129–132

    Google Scholar 

  • Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204

    Article  CAS  Google Scholar 

  • Shabnum S, Wagay MG (2011) Micropropagation of different species of Thymus. J Res Dev 11:71–81

    Google Scholar 

  • Solyman E, Alkownii R (2014) RAPD for assessment of thymes genetic diversity in Palestine. Palestine Tech Univ Res J 2:1–8

    Google Scholar 

  • Stahl-Biskup E, Sáez F (2002) Thyme-the genus Thymus. Taylor & Francis, London, UK, p 75

    Book  Google Scholar 

  • Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109

    Article  CAS  PubMed  Google Scholar 

  • 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 122:573–583

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ, Clustal W (1994) Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tisserat B, Vaughn SF (2001) Essential oils enhanced by ultra-high carbon dioxide levels from Lamiaceae species grown in vitro and in vivo. Plant Cell Rep 20:361–368

    Article  CAS  Google Scholar 

  • Tisserat B, Vaughn SF, Silman R (2002) Influence of modified oxygen and carbon dioxide atmospheres on mint and thyme plant growth, morphogenesis and secondary metabolism in vitro. Plant Cell Rep 20:912–916

    Article  CAS  Google Scholar 

  • Tsoktouridis G, Koutinas N, Osmantzikidis I, Giannakoula A, Roubos K, Sotiropoulos T, Podbeltseva L, Maloupa E, Krigas N (2014) Phylogenetic analysis of 15 eucalyptus species through amplification of the intergenic space of the clpP gene of chloroplast DNA. J Environ Protect Ecol 15:184–193

    CAS  Google Scholar 

  • White PR (1943) A handbook of plant tissue culture. The Jacques Catlell Press, Lancaster, PA

    Book  Google Scholar 

  • Yamane K, Kawahara T (2005) Intra-and interspecific phylogenetic relationships among diploid Triticum-Aegilops species (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences. Am J Bot 92:1887–1898

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research project was funded under the Project ‘Research & Technology Development Innovation Projects’-AgroETAK, MIS 453350, in the framework of the Operational Program ‘Human Resources Development’. It was co-funded by the European Social Fund through the National Strategic Reference Framework (Research Funding Program 2007-2013) coordinated by the Hellenic Agricultural Organization – DEMETER (Institute of Plant Breeding and Genetic Resources).

Author information

Author notes

  1. Georgios Tsoktouridis and Nikos Krigas contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Conservation and Evaluation of Native and Floricultural Species-Balkan Botanic Garden of Kroussia, Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization – Demeter, P.O. Box 60458, GR-570 01, Thermi, Thessaloniki, Greece

    Georgios Tsoktouridis, Nikos Krigas, Virginia Sarropoulou, Katerina Papanastasi, Katerina Grigoriadou & Eleni Maloupa

  2. Laboratory of Agronomy, School of Agriculture, Aristotle University, GR-541 24, Thessaloniki, Greece

    S. Kampouropoulou & Georgios Menexes

Authors
  1. Georgios Tsoktouridis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nikos Krigas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virginia Sarropoulou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Kampouropoulou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katerina Papanastasi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katerina Grigoriadou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Georgios Menexes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Eleni Maloupa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Georgios Tsoktouridis.

Additional information

Editor: Bin Tian

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tsoktouridis, G., Krigas, N., Sarropoulou, V. et al. Micropropagation and molecular characterization of Thymus sibthorpii Benth. (Lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern. In Vitro Cell.Dev.Biol.-Plant 55, 647–658 (2019). https://doi.org/10.1007/s11627-019-10000-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-019-10000-y

Keywords

Search

Navigation