Skip to main content
SpringerLink
Log in

Improved Agrobacterium -mediated transformation of sunflower ( Helianthus annuus L.): assessment of macerating enzymes and sonication

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Agrobacterium -mediated transformation of shoot apices of sunflower ( Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (β-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.

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. 1a–e.
Fig. 2a–e.
Fig. 3.
Fig. 4a–e.
Fig. 5
Fig. 6a–g.

Similar content being viewed by others

Abbreviations

BAP :

6-Benzylaminopurine

GA 3 :

Gibberellic acid

GFP :

Green fluorescent protein

GUS :

β-Glucuronidase

IAA :

Indoleacetic acid

nptII :

Neomycin phosphotransferase II

References

  • Alibert B, Lucas O, Le Gall V, Kallerhoff J, Alibert G (1999) Pectolytic enzyme treatment of sunflower explants prior to wounding and cocultivation with Agrobacterium tumefaciens, enhances efficiency of transient β-glucuronidase expression. Physiol Plant 106:232–237

    CAS  Google Scholar 

  • Alibert G, Aslane-Chanabé C, Burrus M (1994) Sunflower tissue and cell cultures and their use in biotechnology. Plant Physiol Biochem 32:31–44

    Google Scholar 

  • Bidney D, Scelonge C, Martich J, Burrus M, Sims L, Huffman G (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol Biol 18:301–313

    Google Scholar 

  • Burrus M, Chanabé C, Alibert G, Bidney D (1991) Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.). Plant Cell Rep 10:161–166

    Google Scholar 

  • Burrus M, Molinier J, Himber C, Hunold R, Bronner R, Rousselin P, Hahne G (1996) Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns. Mol Breed 2:329–338

    CAS  Google Scholar 

  • Dehmer KJ, Friedt W (1997) Development of molecular markers for high oleic acid content in sunflower (Helianthus annuus L.). Indian Crop Prod 7:311–315

    Article  Google Scholar 

  • Doyle JF, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Grayburn WS, Vick BA (1995) Transformation of sunflower (Helianthus annuus L.) following wounding with glass beads. Plant Cell Rep 14:285–289

    Google Scholar 

  • Hahne G (2001) Sunflower. In: Hui YH, Khatchtourians GG, McHughen A, Nip WK, Scorza R (eds) Handbook of transgenic plants. Marcel Dekker, New York, pp 813–833

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  Google Scholar 

  • Kemner JM, Liang X, Nester EW (1997) The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon. J Bacteriol 179:2452–2458

    CAS  PubMed  Google Scholar 

  • Knittel N, Gruber V, Hahne G, Lenee P (1994) Transformation of sunflower (Helianthus annuus L.): a reliable protocol. Plant Cell Rep 14:81–86

    Google Scholar 

  • Köhler R, Zipfel WR, Webb WW, Hanson MR (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11:613–621

    PubMed  Google Scholar 

  • Korell M, Brahm L, Horn R, Friedt W (1996) Interspecific and intergeneric hybridisation in sunflower breeding. I. General breeding aspects. Plant Breed Abstr 66:925–931

    Google Scholar 

  • Molinier J, Hahne G (1997) Improvement of Agrobacterium tumefaciens-mediated transformation of sunflower (Helianthus annuus L.). In: Pech JC, Latché A, Bouzayen M (eds) Plant sciences. INRA, Versailles, pp 219–222

  • Santarém ER, Trick HN, Essig JS, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759

    Article  Google Scholar 

  • Sheng J, Citovsky V (1996) Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell 8:1699–1710

    Google Scholar 

  • Trick HN, Finer JJ (1997) Sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–337

    CAS  Google Scholar 

  • Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1989) Construction of an intron containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250

    Google Scholar 

  • Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48

    CAS  PubMed  Google Scholar 

  • Weber S, Horn R, Friedt W (2000) High regeneration potential in vitro of sunflower (Helianthus annuus L.) lines derived from interspecific hybridization. Euphytica 116:271–280

    Article  Google Scholar 

  • Weber S, Zarhloul MK, Friedt W (2001) Modification of oilseed quality by genetic transformation. In: Esser K, Kadereit JW, Lüttge U, Runge M (eds) Progress in botany, vol 62. Springer, Berlin Heidelberg New York, pp 140–174

Download references

Acknowledgements

The financial support of the European Community (contract no. AIR-3-CT94-2003) and the Deutsche Forschungsgemeinschaft, Bonn (Project FR-682/8-1), is gratefully acknowledged. We wish to thank Sandra Köhl, Silke Schreiner and Daniela Bernhardt for excellent technical assistance. We like to thank Norbert Martini for providing the construct pBds10FatB3.

Author information

Author notes

  1. S. Weber

    Present address: Plant Science Sweden AB, 26831, Svalöv, Sweden

Authors and Affiliations

  1. Institut für Pflanzenbau und Pflanzenzüchtung I, LS Pflanzenzüchtung, Justus-Liebig-Universität, Heinrich-Buff-Ring 26–32, 35392 , Giessen, Germany

    S. Weber, W. Friedt, N. Landes & R. Horn

  2. Institut de Biologie Moléculaire des Plantes, 12 rue du Rue Général Zimmer, 67084, Strasbourg, France

    J. Molinier, C. Himber, P. Rousselin & G. Hahne

Authors
  1. S. Weber
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. Friedt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. N. Landes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Molinier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Himber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. Rousselin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Hahne
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R. Horn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Horn.

Additional information

Communicated by K. Glimelius

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weber, S., Friedt, W., Landes, N. et al. Improved Agrobacterium -mediated transformation of sunflower ( Helianthus annuus L.): assessment of macerating enzymes and sonication. Plant Cell Rep 21, 475–482 (2003). https://doi.org/10.1007/s00299-002-0548-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-002-0548-7

Keywords

Search

Navigation