Aug 26, 2024
Pollen germination on wheat stigmas
- 1John Innes Centre
- Uauy Lab
Protocol Citation: Marina Millán Blánquez 2024. Pollen germination on wheat stigmas . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy42ol8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 23, 2024
Last Modified: August 26, 2024
Protocol Integer ID: 106332
Keywords: pollen germination, stigma, wheat, aniline blue
Abstract
Aniline blue staining of pollen tube growth on the wheat stigma and through the style 4 h and 30 min after pollination.
Hand pollination
Hand pollination
Please refer to wheat-training.com for detailed explanations on how to perform hand pollinations. Link to pdf: https://www.wheat-training.com/wp-content/uploads/Wheat_growth/pdfs/How-to-cross-wheat-pdf.pdf
Carpel dissection and fixation
Carpel dissection and fixation
Using a pair of tweezers, dissect carpels 4.5 h after pollination to allow sufficient time for pollen tube
emergence.
Store samples in a fixative solution of 95% ethanol and absolute acetic acid (75% v/v) and kept at 4 °C until sample preparation for fluorescence microscopy.
Aniline blue staining of pollinated stigmas
Aniline blue staining of pollinated stigmas
On the day of sampling, prepare a solution of 0.1% aniline blue in 0.1 M K3PO4. You can prepare a stock solution of 1% aniline blue dissolved in 1x PBS. The stock solution should be kept in the fridge at
4 °C. Use tin foil to avoid exposure to light.
Wash fixed samples three times for 5 minutes in sterile water and transferred to 0.1% aniline blue
solution and kept overnight at 4 °C. Use tin foil to avoid exposure to light.
Without washing the samples, dissect out the ovary using a sharp razor blade. Try not to damage the stigma. Leave the remaining stigmatic tissue to dry at 45 °C in a hot plate for a few minutes until most of the aniline blue solution is evaporated. Cover the hot plate with an opaque lid to avoid exposure to light.
Use Vectashield (catalogue No. H-1000-1, 2BSCIENTIFIC LTD) as an antifade mounting media to
preserve fluorescence.
Happy microscopy.