Mar 07, 2025
Version 1
Acetolysis - Bryant et al. 2025 V.1
- 1University of Cincinnati
Protocol Citation: Jacob Bryant 2025. Acetolysis - Bryant et al. 2025. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8o4nv5b/v3
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: October 22, 2024
Last Modified: March 07, 2025
Protocol Integer ID: 110559
Abstract
Bryant et al. 2024 Acetolysis Procedure. Pollen was prepared by a slightly modified standard acetolysis method (Erdtman, 1971; Radford et al. 1974; Pollen Processing Reference Manual 2014; Jones, 2014).
Sample Prep:
Sample Prep:
Anthers are placed into a 1.5mL tube and crushed.
Add glacial acetic acid and stir.
Centrifuge for 3min at 3700rpm to allow pollen to form a pellet at the bottom of the tube. If pollen sticks to the side of the tubes, rotate the tubes 180 degrees and re-centrifuge for 30s.
Draw out liquid with a pipette without disturbing the pellet. If pellet is disturbed, returnthe liquid to the tube, re-centrifuge and draw out again.
Acetolysis:
Acetolysis:
Add 300uL 9:1 acetic anhydride: sulfuric acid acetolysis solution to samples and immediately transfer to heat block.
Incubate at 90°C for about 3-10min with caps open. Finished samples should appear brown in color
Glacial Acetic Acid Wash:
Glacial Acetic Acid Wash:
Add 300uL of glacial acetic acid to all heated samples to stop the reaction.
Cap then vortex thoroughly. Rinse all glassware with glacial acetic acid and then water.
Spin at 3700 rpm for 3min
Draw out liquid using pipette.
Repeat glacial acetic acid wash one or two additional times
Water Wash:
Water Wash:
Add 300uL of water using a clean pipette tip.
Cap samples, vortex then spin again.
Draw water down to pollen pellet.
Repeat water wash one more time.
Ethanol Wash:
Ethanol Wash:
Add 300uL of 50% ethanol to each sample.
Vortex thoroughly.
Spin at 3700rpm 3min.
Draw liquid out with pipette down to the pollen pellet.
Repeat procedure with 70% and 95% ethanol.
Add Glycerin:
Add Glycerin:
Add 3 drops of 1:1 glycerol/water to pollen residue.
Place on heat block at 25°C and leave overnight.
Mix samples, then mount on glass slide for viewing.
Adopted From:
Adopted From:
Erdtman G (1960) The acetolysis method- a revised description. Svensk Botanisk
Tidskrift 54: 561–564.
Jones GD (2014) Pollen analyses for pollination research, acetolysis. Journal of
Pollination Ecology 13: 203–217.
Park KB, Kim JS, Chung GY, Kim JH, Son DC, Jang CG (2022) Clematis pseudotubulosa (Ranunculaceae), a new species from Korea. Korean Journal of Plant Taxonomy 52: 35–44.
Pollen processing reference manual (2014) Florida Institute of Technology.