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Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen

  • Cell Biology and Morphogenesis
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Abstract

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical.

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Abbreviations

ATP:

Adenosine tris-phosphate

CCD:

Charged coupled device

EGTA:

Ethylene glycol tetraacetic acid

FDA:

Fluorescein diacetate

References

  • Becker JD, Boavida LC, Carneiro J, Haury M, Feijó JA (2003) Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome. Plant Physiol 133:713–725

    Article  PubMed  CAS  Google Scholar 

  • Blevins DG, Lukaszewski KM (1998) Boron in plant structure and function. Annu Rev Plant Physiol Plant Mol Biol 49:481–500

    Article  PubMed  CAS  Google Scholar 

  • Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52:570–582

    Article  PubMed  CAS  Google Scholar 

  • Brewbaker J, Kwack B (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865

    Article  CAS  Google Scholar 

  • Chebli Y, Geitmann A (2007) Mechanical principles governing pollen tube growth. Funct Plant Sci Biotechnol 1:232–245

    Google Scholar 

  • Elleman C, Franklin-Tong V, Dickinson H (1992) Pollination in species with dry stigmas: the nature of the early stigmatic response and the pathway taken by pollen tubes. New Phytol 121:413–424

    Article  Google Scholar 

  • Fan LM, Wang YF, Wang H, Wu WH (2001) In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. J Exp Bot 52:1603–1614

    Article  PubMed  CAS  Google Scholar 

  • Feijó JA, Malhó R, Obermeyer G (1995) Ion dynamics and its possible role during in-vitro pollen germination and tube growth. Protoplasma 187:155–167

    Article  Google Scholar 

  • Geitmann A, Cresti M (1998) Ca2+ channels control the rapid expansions in pulsating growth of Petunia hybrida pollen tubes. J Plant Physiol 152:439–447

    CAS  Google Scholar 

  • Guyon VN, Astwood JD, Garner EC, Dunker AK, Taylor LP (2000) Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in Petunia. Plant Physiol 123:699–710

    Article  PubMed  CAS  Google Scholar 

  • Holdaway-Clarke TL, Weddle NM, Kim S, Robi A, Parris C, Kunkel JG, Hepler PK (2003) Effect of extracellular calcium, pH and borate on growth oscillations in Lilium formosanum pollen tubes. J Exp Bot 54:65–72

    Article  PubMed  CAS  Google Scholar 

  • Johnson-Brousseau SA, McCormick S (2004) A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J 39:761–775

    Article  PubMed  CAS  Google Scholar 

  • Li H, Lin YK, Heath RM, Zhu MX, Yang ZB (1999) Control of pollen tube tip growth by a pop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11:1731–1742

    Article  PubMed  CAS  Google Scholar 

  • Mouline K, Very AA, Gaymard F, Boucherez J, Pilot G, Devic M, Bouchez D, Thibaud JB, Sentenac H (2002) Pollen tube development and competitive ability are impaired by disruption of a shaker K+ channel in Arabidopsis. Genes Dev 16:339–350

    Article  PubMed  CAS  Google Scholar 

  • Parre E, Geitmann A (2005) More than a leak sealant. The mechanical properties of callose in pollen tubes. Plant Physiol 137:274–286

    Article  PubMed  CAS  Google Scholar 

  • Picton JM, Steer MW (1983) Evidence for the role of Ca2+ ions in tip extension in pollen tubes. Protoplasma 115:11–17

    Article  CAS  Google Scholar 

  • Schnurer J, Rosswall T (1982) Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. Appl Environ Microbiol 43:1256–1261

    PubMed  CAS  Google Scholar 

  • Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767

    Article  PubMed  CAS  Google Scholar 

  • Steer MW, Steer JM (1989) Pollen tube tip growth. New Phytol 111:323–358

    Article  Google Scholar 

  • Taylor LP, Hepler PK (1997) Pollen germination and tube growth. Annu Rev Plant Physiol Plant Mol Biol 48:461–491

    Article  PubMed  CAS  Google Scholar 

  • The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

    Article  Google Scholar 

  • Tuinstra MR, Wedel J (2000) Estimation of pollen viability in grain Sorghum. Crop Sci 40:968–970

    Article  Google Scholar 

  • Tupý J, Říhová L (1984) Changes and growth effect of pH in pollen tube culture. J Plant Physiol 115:1–10

    Google Scholar 

  • Van Der Woude WJ, Morre DJ, Bracker CE (1971) Isolation and characterization of secretory vesicles in germinated pollen of Lilium longiflorum. J Cell Sci 8:331–351

    Google Scholar 

  • Wang QL, Lu LD, Wu XQ, Li YQ, Lin JX (2003) Boron influences pollen germination and pollen tube growth in Picea meyeri. Tree Physiol 23:345–351

    PubMed  CAS  Google Scholar 

  • Yamamoto Y, Nishimura M, Hara-Nishimura I, Noguchi T (2003) Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana. Plant Cell Physiol 44:1192–1201

    Article  PubMed  CAS  Google Scholar 

  • Yokota E, Shimmen T (1994) Isolation and characterization of plant myosin from pollen tubes of lily. Protoplasma 177:153–162

    Article  CAS  Google Scholar 

  • Zinkl GM, Preuss D (2000) Dissecting Arabidopsis pollen-stigma interactions reveals novel mechanisms that confer mating specificity. Ann Bot 85:15–21

    Article  Google Scholar 

Download references

Acknowledgments

Research in the Geitmann lab is supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) and the Human Frontier Science Program (HFSP).

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Authors and Affiliations

  1. Département de Sciences Biologiques, Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 rue Sherbrooke est, Montreal, QC, H1X 2B2, Canada

    Firas Bou Daher, Youssef Chebli & Anja Geitmann

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  1. Firas Bou Daher
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  2. Youssef Chebli
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  3. Anja Geitmann
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Corresponding author

Correspondence to Anja Geitmann.

Additional information

Communicated by F. Brandizzi.

F. Bou Daher and Y. Chebli contributed equally to this study.

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Bou Daher, F., Chebli, Y. & Geitmann, A. Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen. Plant Cell Rep 28, 347–357 (2009). https://doi.org/10.1007/s00299-008-0647-1

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  • DOI: https://doi.org/10.1007/s00299-008-0647-1

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