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[Bio101] Arabidopsis Pollen Tube Aniline Blue Staining   

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Abstract

The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception.

Materials and Reagents

  1. Stock solutions:
    1. Acetic acid
    2. Ethanol
    3. NaOH
    4. K2HPO4
    5. KH2PO4
    6. Aniline blue (Thermo Fisher Scientific)
    7. Glycerol
  2. Working solutions:
    1. 10% acetic acid in EtOH (fixative)
    2. 0.01% aniline blue in KPO4 buffer (dye)
    3. KPO4 buffer made with 50% glycerol (mounting media)
    4. KPO4 buffer (see Recipes)

Equipment

  1. Microscope with UV light

Procedure

  1. Submerge pistil tissue in 250 µl acetic acid and fix it for 1.5 h or more in an Eppendorf tube. Tissue can be fixed overnight if necessary.
  2. Soften tissue by submerging it in 1 M NaOH overnight.
  3. Wash 3 times with KPO4 buffer (tissue is fragile at this stage).
  4. Stain with 200 µl aniline blue for 5-10 min or as long as 10 h.
  5. Transfer to a slide, add mounting media and observe under UV. Squash if necessary.

Recipes

  1. 50 mM KPO4 buffer (pH 7.5)
    4.17 ml 1 M K2HPO4
    0.83 ml 1 M KH2PO4
    995 ml H2O

References

  1. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.
  2. Modified from the online protocol on Dr. Daphne Preusss’s lab (Univ of Chicago).
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Lu, Y. (2011). Arabidopsis Pollen Tube Aniline Blue Staining. Bio-protocol Bio101: e88. DOI: 10.21769/BioProtoc.88.
Q&A

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3/26/2015 6:02:52 AM Reply
3/10/2015 12:17:09 AM Reply

hi , can i use this method for observation of pollen tube growth "in embryo sac" ?

thanks
4/22/2012 12:06:39 AM Reply
Yuanqing Lin
7/11/2011 2:20:50 PM Reply
bio-protocol

Aniline blue stains callose, which is a major chemical component of the pollen tube cell wall. That makes it a good dye to visualize pollen tube growing in vivo.

7/11/2011 2:21:17 PM