Skip to main content
SpringerLink
Log in

In vitro induction of tetraploids in ornamental Ranunculus

  • Published:
Euphytica Aims and scope Submit manuscript

Abstract

This study investigates the capacity of the antimitotic agents colchicine, oryzalin and trifluralin for inducing polyploidisation of Ranunculus asiaticus ‘Alfa’ in vitro shoots. Flow cytometry was used to evaluate the optimal concentration of each antimitotic agent for polyploidisation. Trifluralin at a concentration of 2 μM resulted in the highest percentage of polyploidisation (27.5%), followed by a colchicine treatment of 200 μM, which induced 23.3% of polyploids. For oryzalin the highest percentage was achieved using a concentration of 1 μM. Different exposure periods were tested and turned out to be an important factor. The maximal exposure period tested (10 weeks) resulted in a significant increase in polyploidisation by oryzalin and trifluralin. In contrast, for colchicine (100 μM) exposure times of either 16 or 24 h did not significantly influence polyploidisation. Additionally the effect of the antimitotic agents on the viability was analysed. For colchicine no significant effect on the survival rate was observed, for trifluralin only a concentration of 10 μM affected viability whereas for oryzalin, concentration as well as exposure period were significant parameters. Flow cytometric data were confirmed by counting chromosomes in root tip cells.

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

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

References

  • Allum JF, Bringloe DH, Roberts AV (2007) Chromosome doubling in a Rosa rugosa Thunb. hybrid by exposure of in vitro nodes to oryzalin: the effects of node length, oryzalin concentration and exposure time. Plant Cell Rep 26:1977–1984. doi:10.1007/s00299-007-0411-y

    Article  PubMed  CAS  Google Scholar 

  • Baack EJ (2004) Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus: Ranunculaceae). Am J Bot 91:1783–1788. doi:10.3732/ajb.91.11.1783

    Article  Google Scholar 

  • Baack EJ (2005) Ecological factors influencing tetraploid establishment in snow buttercup (Ranunculus adoneus, Ranunculaceae): minority cytotype exclusion and barriers to triploid formation. Am J Bot 92:1827–1835. doi:10.3732/ajb.92.11.1827

    Article  Google Scholar 

  • Bajer AS, Molebajer J (1986) Drugs with colchicine-like effects that specifically disassemble plant but not animal microtubules. Ann N Y Acad Sci 466:767–784. doi:10.1111/j.1749-6632.1986.tb38458.x

    Article  PubMed  CAS  Google Scholar 

  • Beruto M, Debergh P (2004) Micropropagation of Ranunculus asiaticus: a review and perspectives. Plant Cell Tissue Organ Cult 77:221–230. doi:10.1023/B:TICU.0000018416.38569.7b

    Article  Google Scholar 

  • Chauvin JE, Label A, Kermarrec MP (2005) In vitro chromosome-doubling in tulip (Tulipa gesneriana L.). J Hortic Sci Biotechnol 80:693–698

    Google Scholar 

  • Cohen D, Yao JL (1996) In vitro chromosome doubling of nine Zantedeschia cultivars. Plant Cell Tissue Organ Cult 47:43–49. doi:10.1007/BF02318964

    Article  Google Scholar 

  • Dart S, Kron P, Mable BK (2004) Characterizing polyploidy in Arabidopsis lyrata using chromosome counts and flow cytometry. Can J Bot 82:185–197. doi:10.1139/b03-134

    Article  Google Scholar 

  • de Nettancourt D (2001) Incompatibility and incongruity in wild and cultivated plants. Springer-Verlag, Berlin

    Google Scholar 

  • Dhooghe E, Grunewald W, Leus L, Van Labeke MC (2009) In vitro polyploidisation of Helleborus species. Euphytica 165:89–95. doi:10.1007/s10681-008-9763-9

    Article  Google Scholar 

  • Dunn BL, Lindstrom JT (2007) Oryzalin-induced chromosome doubling in Buddleja to facilitate interspecific hybridization. HortScience 42:1326–1328

    Google Scholar 

  • Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051. doi:10.1126/science.220.4601.1049

    Article  PubMed  CAS  Google Scholar 

  • Horovitz A (1985) Ranunculus. In: Halevy AH (ed) CRC handbook of flowering, vol IV. CRC Press, Florida, pp 155–161

    Google Scholar 

  • Hugdahl JD, Morejohn LC (1993) Rapid and reversible high-affinity binding of the dinitroaniline herbicide oryzalin to tubulin from Zea mays L. Plant Physiol 102:725–740

    PubMed  CAS  Google Scholar 

  • Kermani MJ, Sarasan V, Roberts AV, Yokoya K, Wentworth J, Sieber VK (2003) Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability. Theor Appl Genet 107:1195–1200. doi:10.1007/s00122-003-1374-1

    Article  PubMed  CAS  Google Scholar 

  • Khosravi P, Kermani MJ, Nematzadeh GA, Bihamta MR, Yokoya K (2008) Role of mitotic inhibitors and genotype on chromosome doubling of Rosa. Euphytica 160:267–275. doi:10.1007/s10681-007-9571-7

    Article  CAS  Google Scholar 

  • Langlet O (1932) Über Chromosomenverhältnisse und Systematik der Ranunculaceae. Svensk Botanisk Tidskrift 26:381–400

    Google Scholar 

  • Lu C, Bridgen MP (1997) Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea. Euphytica 94:75–81. doi:10.1023/A:1002911522748

    Article  Google Scholar 

  • Mable BK (2004) Polyploidy and self-incompatibility: is there an association? New Phytol 162:803–811. doi:10.1111/j.1469-8137.2004.01055.x

    Article  Google Scholar 

  • Mears JA (1980) Chemistry of polyploids. In: Lewis WH (ed) Polyploidy: biological relevance, vol 13. Plenum Press, New York, pp 77–102

    Google Scholar 

  • Meynet J (1993) Ranunculus. In: De Hertogh A, Le Nard M (eds) The physiology of flower bulbs. Elsevier Science Publishers, The Netherlands, pp 603–610

    Google Scholar 

  • Morejohn LC, Bureau TE, Tocchi LP, Fosket DE (1984) Tubulins from different higher-plant species are immunologically nonidentical and bind colchicine differentially. Proc Natl Acad Sci USA 81:1440–1444. doi:10.1073/pnas.81.5.1440

    Article  PubMed  CAS  Google Scholar 

  • Morejohn LC, Bureau TE, Molebajer J, Bajer AS, Fosket DE (1987) Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and inhibits microtubule polymerization in vitro. Planta 172:252–264. doi:10.1007/BF00394595

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  • Ohkawa K (1986) Growth and flowering of Ranunculus asiaticus. Acta Hortic 177:165–177

    Google Scholar 

  • Otto F (1990) DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Darzynkiewicz Z, Crissman HA (eds) Methods in cell biology, vol 33. Academic Press Inc, New York, pp 105–110

    Google Scholar 

  • Paden DW, Meyer MM, Rayburn AL (1990) Doubling chromosomes with colchicine treatment in vitro as determined by chloroplast number in epidermal guard cells. J Am Rhododendron Soc 44:162–167

    Google Scholar 

  • Ro KE, Keener CS, McPheron BA (1997) Molecular phylogenetic study of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships. Mol Phylogenet Evol 8:117–127. doi:10.1006/mpev.1997.0413

    Article  PubMed  CAS  Google Scholar 

  • Smith JB, Bennett MD (1975) DNA variation in the genus Ranunculus. Heredity 35:231–239. doi:10.1038/hdy.1975.87

    Article  Google Scholar 

  • Soltis PS, Soltis DE (2000) The role of genetic and genomic attributes in the success of polyploids. Proc Natl Acad Sci USA 97:7051–7057. doi:10.1073/pnas.97.13.7051

    Article  PubMed  CAS  Google Scholar 

  • Stanton ML, Galen C, Shore J (1997) Population structure along steep environmental gradient: consequences of flowering time and habitat variation in snow buttercup, Ranunculus adoneus. Evolution Int J Org Evolution 51:79–94. doi:10.2307/2410962

    Google Scholar 

  • Stanys V, Weckman A, Staniene G, Duchovskis P (2006) In vitro induction of polyploidy in Japanese quince (Chaenomeles japonica). Plant Cell Tissue Organ Cult 84:263–268. doi:10.1007/s11240-005-9029-3

    Article  CAS  Google Scholar 

  • Stebbins GL (1971) Chromosome evolution in higher plants. Edward Arnold, London

    Google Scholar 

  • Takamura T, Miyajima I (1996) Colchicine induced tetraploids in yellow-flowered cyclamens and their characteristics. Sci Hortic (Amsterdam) 65:305–312. doi:10.1016/0304-4238(96)00896-5

    Article  CAS  Google Scholar 

  • Takamura T, Lim KB, Van Tuyl JM (2002) Effect of a new compound on the mitotic polyploidisation of Lilium longiflorum and oriental hybrid lilies. Acta Hortic 572:37–40

    CAS  Google Scholar 

  • Tal M (1980) Physiology of polyploids. In: Lewis WH (ed) Polyploidy: biological relevance, vol 13. Plenum Press, New York, pp 61–76

    Google Scholar 

  • Tamura M (1993) Ranunculaceae. In: Kubitski K, Rohwer JG, Bittrich V (eds) The families and genera of vascular plants: flowering plants—dicotyledons, vol II. Springer-Verlag, Berlin, pp 563–583

    Google Scholar 

  • Väinölä A (2000) Polyploidization and early screening of Rhododendron hybrids. Euphytica 112:239–244. doi:10.1023/A:1003994800440

    Article  Google Scholar 

  • Wang ZF, Ren Y (2008) Ovule morphogenesis in Ranunculaceae and its systematic significance. Ann Bot (Lond) 101:447–462. doi:10.1093/aob/mcm298

    Article  Google Scholar 

  • Wang W, Li RQ, Chen ZD (2005) Systematic position of Asteropyrum (Ranunculaceae) inferred from chloroplast and nuclear sequences. Plant Syst Evol 255:41–54. doi:10.1007/s00606-005-0339-z

    Article  CAS  Google Scholar 

  • Yamaguchi M (1989) Basic studies on the flower color breeding of carnations (Dianthus caryophyllus L.). Bull Fac Hort Minamikyusyu Univ 19:1–79

    Google Scholar 

  • Zhang Z, Dai H, Xiao M, Liu X (2008) In vitro induction of tetraploids in Phlox subulata L. Euphytica 159:59–65. doi:10.1007/s10681-007-9457-8

    Article  Google Scholar 

  • Zlesak DC, Thill CA, Anderson NO (2005) Trifluralin-mediated polyploidization of Rosa chinensis minima (Sims) Voss seedlings. Euphytica 141:281–290. doi:10.1007/s10681-005-7512-x

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank H. Carlier for the flow cytometric measurements. Special thanks also go to T. Versluys for the technical supports and to IRF (Istituto Regionale per la Floricoltura, Sanremo—Italy) especially to M. Beruto for the supply of the high quality in vitro plant material.

Author information

Authors and Affiliations

  1. Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000, Ghent, Belgium

    Emmy Dhooghe, Sylvie Denis, Dirk Reheul & Marie-Christine Van Labeke

  2. Plant Unit—Applied Genetics and Breeding, Institute for Agricultural and Fisheries Research, Caritasstraat 21, 9090, Melle, Belgium

    Tom Eeckhaut

Authors
  1. Emmy Dhooghe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sylvie Denis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tom Eeckhaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dirk Reheul
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marie-Christine Van Labeke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Emmy Dhooghe.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dhooghe, E., Denis, S., Eeckhaut, T. et al. In vitro induction of tetraploids in ornamental Ranunculus . Euphytica 168, 33–40 (2009). https://doi.org/10.1007/s10681-008-9876-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10681-008-9876-1

Keywords

Search

Navigation