Sep 03, 2024
Heptane preparation of Annonaceae pollen
- Couvreur LP Thomas1,
- Tanawat Chaowasku2,
- Raymond W. J. M. van der Ham3
- 1Institut de Recherche pour le Développent (IRD);
- 2Herbarium, Department of Biology, Faculty of Science, Chiang Mai University, 239 Huay Kaew Rd., Chiang Mai 50200, Thailand;
- 3Naturalis Biodiversity Center | NCB · Department of Botany
Protocol Citation: Couvreur LP Thomas, Tanawat Chaowasku, Raymond W. J. M. van der Ham 2024. Heptane preparation of Annonaceae pollen. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r6r5g1b/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: March 29, 2024
Last Modified: September 03, 2024
Protocol Integer ID: 97529
Keywords: heptane, Annonaceae, pollen
Abstract
The protocol used to study Annonaceae pollen taken from herbarium dried specimens or in field alcohol conserved material. The protocol deviates from previous versions by using Heptane instead of n-Hexane.
Guidelines
This protocol uses Heptane, which is a highly volatil solvent and all steps must be done under a fune cabinet.
Materials
Heptane
Alcohol 100%
Glass vials
Gloves
Piptette
Fume cabinet
Safety warnings
Use a fume cabinet.
Use gloves.
Careful for contamination between samples.
Ethics statement
Make sure you have the requested permits to sample herbarium material from the herbarium curator.
Sample material
Sample material
10m
10m
Use a stereo microscope to collect anthers at anthesis of flowers from herbarium specimen or conserved in alcohol (spirit material). Depending on the size of the anthers take 3-10. Place the anthers in a glass vial.
Example of a stereo microscope
5m
Crush the anthers with a glass rod. Make sure to clean the rod in between crushed to avoid contamination.
Crushed anthers
5m
If you have alcohol preserved material continue; for herbarium dried material jump to Step 6.
Alcohol preserved material
Alcohol preserved material
10m
10m
Wash anthers in alcohol 100% for 00:05:00 . Remove excess alcohol if needed.
Washing of alcohol preserved anthers/pollen with 100% alcohol.
5m
Place vials in fume cabinet.
Wash again but this time with alcohol 100% + Hepatne in a 1/1 mix for 00:05:00 .
5m
Herbarium preserved material
Herbarium preserved material
19m
19m
Start here with herbarium dried material, and continue here with alcohol preserved material. Place vials in fume cabinet.
Add 1 mL of Heptane to each vial, swirl the solution a bit and then leave for 00:01:00 .
Heptane solvent ready for use
1m
Remove most of the Heptane carefully with a pipette. Try to keep the pollen material on the bottom of the vial; take a clean pipette for each sample to avoid contamination.
2m
Repeat steps 6 and 7 twice. The samples must go through three rounds of washing.
6m
After the third step of Heptane, try to pipette off as much of the solution as possible without removing pollen material. Let the remaining Heptane evaporate so the sample becomes dry. You can recheck to make sure your sample still has pollen under the stereo microscope.
10m
Transfer the material to labelled tubes for microscope viewing.
1m
Protocol references
- Chaowasku T, Mols J, van der Ham RWJM (2008) Pollen morphology of Miliusa and relatives (Annonaceae). Grana 47: 175–184. https://doi.org/10.1080/00173130802202305
- Couvreur TLP, Botermans M, van Heuven BJ, Van der Ham RWJM (2008) Pollen morphology within the Monodora clade, a diverse group of five African Annonaceae genera. Grana 47: 185–210. https://doi.org/10.1080/00173130802256913
- Van der Ham RWJM (1990) Nephelieae pollen (Sapindaceae): form, function, and evolution. Natl. Herb. Neerl., Leiden.