Antisense Oligodeoxynucleotide-Mediated Gene Knockdown in Pollen Tubes

Part of the Methods in Molecular Biology book series (MIMB, volume 1080)


Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy recently emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen tubes. Here, we describe the protocol for the successful employment of AODN technique in growing tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the advantages and drawbacks of this method.

Key words

Antisense oligodeoxynucleotide AODN Pollen tube Gene knockdown Tip growth 



Pollen tube research in the Žárský lab is supported by the Czech Grant Agency grant GACR 13-19073S to M. P.


  1. 1.
    Cole RA, Fowler JE (2006) Polarized growth: maintaining focus on the tip. Curr Opin Plant Biol 9:579–588PubMedCrossRefGoogle Scholar
  2. 2.
    Potocký M, Eliáš M, Profotová B et al (2003) Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth. Planta 217:122–130PubMedGoogle Scholar
  3. 3.
    Potocký M, Jones MA, Bezvoda R et al (2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174:742–751PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang Y, McCormick S (2010) The regulation of vesicle trafficking by small GTPases and phospholipids during pollen tube growth. Sex Plant Reprod 23:87–93PubMedCrossRefGoogle Scholar
  5. 5.
    Watts JK, Corey DR (2012) Silencing disease genes in the laboratory and the clinic. J Pathol 226:365–379PubMedCrossRefGoogle Scholar
  6. 6.
    Sun C, Höglund A-S, Olsson H et al (2005) Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling. Plant J 44:128–138PubMedCrossRefGoogle Scholar
  7. 7.
    Pleskot R, Potocký M, Pejchar P et al (2010) Mutual regulation of plant phospholipase D and the actin cytoskeleton. Plant J 62:494–507PubMedCrossRefGoogle Scholar
  8. 8.
    Walder RY, Walder JA (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA 85:5011–5015PubMedCrossRefGoogle Scholar
  9. 9.
    Moulton HM, Nelson MH, Hatlevig SA et al (2004) Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 15:290–299PubMedCrossRefGoogle Scholar
  10. 10.
    Okuda S, Tsutsui H, Shiina K et al (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458:357–361PubMedCrossRefGoogle Scholar
  11. 11.
    Tsutsumi N, Kanayama K, Tano S (1992) Suppression of alpha-amylase gene expression by antisense oligodeoxynucleotide in barley cultured aleurone layers. Jinrui Idengaku Zasshi 67:147–154Google Scholar
  12. 12.
    Estruch JJ, Kadwell S, Merlin E et al (1994) Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 91:8837–8841PubMedCrossRefGoogle Scholar
  13. 13.
    Moutinho A, Hussey PJ, Trewavas AJ et al (2001) cAMP acts as a second messenger in pollen tube growth and reorientation. Proc Natl Acad Sci USA 98:10481–10486PubMedCrossRefGoogle Scholar
  14. 14.
    Camacho L, Malhó R (2003) Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases. J Exp Bot 54:83–92PubMedCrossRefGoogle Scholar
  15. 15.
    Sun C, Ridderstråle K, Höglund A-S et al (2007) Sweet delivery - sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells. Plant J 52:1192–1198PubMedCrossRefGoogle Scholar
  16. 16.
    de Graaf BHJ, Rudd JJ, Wheeler MJ et al (2006) Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen. Nature 444:490–493PubMedCrossRefGoogle Scholar
  17. 17.
    Pleskot R, Pejchar P, Bezvoda R et al (2012) Turnover of phosphatidic acid through distinct signaling pathways affects multiple aspects of pollen tube growth in tobacco. Front Plant Sci 3:54PubMedCrossRefGoogle Scholar
  18. 18.
    Potocký M, Pejchar P, Gutkowska M et al (2012) NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases. J Plant Physiol 169:1654–1663PubMedCrossRefGoogle Scholar
  19. 19.
    Hafidh S, Breznenová K, Růžička P et al (2012) Comprehensive analysis of tobacco pollen transcriptome unveils common pathways in polar cell expansion and underlying heterochronic shift during spermatogenesis. BMC Plant Biol 12:24PubMedCrossRefGoogle Scholar
  20. 20.
    Read SM, Clarke AE, Bacic A (1993) Stimulation of growth of cultured Nicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts. Protoplasma 177:1–14CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  1. 1.Department of Experimental Plant Biology, Faculty of ScienceCharles University and Institute of Experimental Botany, Academy of Sciences of the Czech RepublicPragueCzech Republic
  2. 2.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPragueCzech Republic

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