Stigma staining

Protocol

Author

Adrienne Nicotra

Overview

This protocol makes use of a basic fuchsin dye to stain collected pollen grains to allow for counts of stigma pollen loads. It also outlines a style preparation technique for counting pollen tubes.

Background

This protocol makes use of a basic fuchsin dye to stain collected pollen grains. The fuchsin dye is deposited on the outer wall of the pollen grains, enhancing the surface characteristics necessary for identification. It does not usually stain fungal spores or other debris. The stain is carried in Calberla’s solution.

Materials/Equipment

  • Flowers from species of interest
  • Scalpel
  • Forceps
  • Vial
  • 60% bleach solution
  • Watch glass
  • Distilled H2O
  • 50% glycerol (from supplier, or made using recipe below)
  • Slides and coverslips
  • Compound microscope

For Calberla’s solution

  • Glycerin
  • 95% ethyl alcohol
  • Saturated aqueous basic fuchsin
  • Glycerin jelly.

For Glycerine jelly

  • Fine gelatine
  • Paraffin oven
  • Glycerol
  • Cresol
  • Muslin
  • Small balsam bottle

Procedure

Stigma pollen load – preparation to mounted slide stage

  1. Cut stigma off style, making a clean cut at an acute angle (so that when it comes to doing pollen tube counts on the style you can tell the top from the bottom).
  2. Place stigmas in a vial of 60% bleach solution for 40-50 min. The aim behind bleaching is to reduce the colour to a faint tea stain appearance, not all white. Some parts may bleach white but if all specimens are all white, then the bleaching needs to be adjusted lower. Some will bleach more readily than others.
  3. Rinse by placing bleached stigmas in watch glass of distilled H2O for ~10 min
  4. Place stigmas in Calberla’s solution (see recipe below) , stain for 6 min (run preliminary test to determine if grains are staining strongly – if not, increase the stain time)
  5. Mount stigmas in drop of 50% glycerol, cover with a coverslip
  6. Count pollen grains under the microscope

Making Calberla’s solution

  • Modified recipe: Combine 5 ml glycerin, 10 ml 95% ethyl alcohol, 15 ml distilled water, 2 or 3 drops saturated aqueous basic fuchsin, and 2 or 3 drops of melted glycerin jelly.

”Making Glycerol/Glycerine jelly (which can also be purchased from scientific suppliers)

  1. Soak 5 g of fine gelatine for 1 hr in 25 ml of cold distilled water.
  2. Cover the vessel and place it in the paraffin oven and stir occasionally until the gelatine has dissolved.
  3. Meanwhile, mix 35 ml of glycerol, 40 ml of water and 0.25 ml of cresol, and leave the resultant mixture in the paraffin oven.
  4. Once the gelatine has dissolved, mix the two fluids thoroughly and while still hot (e.g. in a large incubator), filter through a piece of muslin.
  5. When cool the medium will set to a gel. However while it is still warm, pour a small amount into a small balsam bottle so it is readily available to be melted in the paraffin oven as required.

(Glycerine jelly recipe modified from Baker (1966))

Pollen tube – style preparation to mounted slide stage

  1. Locate style in microtube, rinse in water for ~2 min
  2. Allow tissue to clear for ca. 2 1/2 hrs
  3. Transfer to stain – setup 5 min plus n samples x 20 seconds (ca 15 min)
  4. Leave to stain overnight
  5. Mount on slide, label and place a coverslip over. Inspect base end (looking for angle of the cut from stigma removal), mark, squash slide. ~2 min
  6. Make pollen tube counts under the microscope (~1 min per specimen)

Notes and troubleshooting tips

This protocol has been used effectively on flowers of the following species, with minor modifications for each: Dianella revoluta

  • Recover style from closed flower under dissecting microscope (~3 min)
  • Remove stigma, rinse, label slide, squash (~3 min)
  • Stain in Carberla’s solution (4 min 30 sec)

Senna artemisioides

  • Cut off close to stigma, with as little style as possible (~1 min)
  • Stain for 6 minutes

Eremophila glabra

  • As for Senna but its not so critical to cut the style as short as possible

Literature references

Baker, J. (1966) Chapter VII – Mounting, of Cytological Technique: The Principles and Practice of Methods used to determine the Structure of the Metazoan Cell, Methuen & Co. Ltd: London & John Wiley & Sons, Inc: New York. p 172-173

Duncan DH, Nicotra AB, Cunningham SA (2004) High self-pollen transfer and low fruit set in buzz-pollinated Dianella revoluta (Phormiaceae). Australian Journal of Botany 52(2), 185-193.

 

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