FAST PROTOCOL FOR TETRAZOLIUM TEST
IN ORCHID SEEDS
(Machado-Neto, NB; Custódio, CC; Hosomi, ST; Seaton, PT; Marks, TR)

1. Weigh 3-5mg of seeds
2. Place in 1.5ml microtube
3. Add 1.5ml of 10% sucrose solution
4. Leave the tube at room temperature, or at 25ºC, for 24 h
5. Draw off the solution using either a Pasteur pipette or a micropipette
6. Add 1ml of Tetrazolium (TZ) solution (1%)
7. Place in a water bath at 40ºC for 24h
8. Change the TZ solution with water using a micropipette. The counting can be carried out immediately or after storing in a refrigerator until a more convenient time.
9. Place the solution containing the seeds on a microscope slide;
10. Place the slide on a flat bed scanner and scan the image at 3600dpi, placing a clear plastic rule by the side of the slide to act as a scale. The inner face of the scanner cover must be covered by a blue sheet for better contrast.
11. Usually the whole slide is read on the computer screen, but alternatively it may be split into sections of known area.

Tetrazolium prepared in phosphate buffer

Where it is important to maintain the pH (6.5 - 7.5) for optimal activity, the tetrazolium (TZ) can be dissolved in a phosphate buffer.  To prepare one litre:

1. Dissolve 3.63g of potassium dihydrogen orthophosphate (KH2PO4) in 400 ml of deionised water.
2. Dissolve 7.13g disodium hydrogen orthophosphate dihydrate (Na2HPO4) in 600 ml of deionised water.
3. When these two solutions are completely dissolved, mix together then add and dissolve TZ at the required concentration  e.g. for a 1% w/v solution add 1g TZ.
4. Store the solution in a glass bottle, exclude any light and store at 4°C for no longer than three months

Note: Any reagent that develops a pink colour should be discarded.

1. Seeds may be washed with distilled water.
2. Weigh 1g of 2,3,5 Triphenyl tetrazolium chloride and dissolve it in 100 ml of water.
It must be stored under refrigeration in an amber flask.
3. Shorter times could be used see Hosomi et al. (2011) SST 39(1)178-189

Close-up of seeds of seeds of Orchis sancta showing dark red embryos of viable seeds. Photo Khaled Abulaila

Viable (deep red embryos) and non-viable (pale embryos) seeds of
Orchis papilionaceae. Note the grid lines to assist counting of viable and
non-viable seeds. Photo Khaled Abulaila.