Table 3.

A standard operating procedure for the spectrophotometric assay to test the viability of seed batches: using minimum of three technical replicates of barley (Hordeum vulgare cv. optic) seed as a model.

1. Place the seeds between water saturated tissue paper to imbibe over-night (ca. 16 h) in a sealed container.*

2. Remove any seeds with protruded shoot-born roots.

3. Dry the seeds and transfer a fixed weight (7.5 g, ca. 100 seeds), to a 50 mL container.

4. Bleach/sterilise the seeds by soaking in 7 mL 3% hydrogen peroxide (Sigma-Aldrich, #H1009) for 10 min.

5. Wash 2 times with 20 mL of sterile distilled water.

6. Dry the seeds with tissue paper before grinding in blender for 1 min (James Martin by Wahl ZX595 Mini Grinder, 150 W)

7. Transfer all the seed flour to a fresh 50 mL tube.*

8. Add 15 mL of TTC (Sigma-Aldrich, #T8877) in 1% [w/v] with 5mM potassium phosphate buffer, ph 7.2 (Peters, 2000).

9. Homogenise by vortexing for 15 s.

10. Incubate for 4 h in darkness at room temperature (21°C).*

11. Centrifuge at 5,100 rpm for 5 min. (Sigma 4K-15) and remove the supernatanta.

12. Suspend residue in 20 mL sterile distilled water and re-centrifuge (as 11); repeat this step.

13. Transfer the residue to filter-paper (Whatmann No.3) on a Buchner-funnelb.

14. Remove excess moisture from the residue under vacuum.

15. Place the entire residue into a sterile container to dry over-night (21°C).

16. Grind the whole dried residue to a powder using a mortar and pestle.

17. Re-grind the residue after the addition of liquid N2c.

18. Dry at 30°C 10 min. and decant the fine powder to new 50 mL centrifuge tubes.

19. Add 7 mL of 10% TCA (Sigma-Aldrich, #T6399) [w/v] /methanol and vortex 3 × 30 s.*

20. Incubate the samples overnight (15 h) at 30°C.

21. Centrifuge 15 min. at 5,100 rpm at room temperature (Sigma 4K-15).

22. Remove 2 mL of the extract and transfer to microfuge tube.

23. Centrifuge the microfuge tube for 30 min. at 14,680 rpm at room temperature (Sigma 1-15K).

24. Transfer 1.5 mL of the supernatant to fresh 1.5 mL microfuge tubed.

OPTICAL DENSITY (OD) DETERMINATION

25. Re-centrifuge 1.5 mL extract for 20 min. at 14,680 rpm at room temperature (Sigma 1-15K).

26. Dispense 300 μl (extract, controls, blanks), to separate wells of flat-bottomed ELISA plate (Nunc MaxiSorp®, manufacturers code 439454).

27. Read the optical density of each at 484 nm (using at ELx800™ Absorbance Reader, BioTek® Inc.).

28. Correct the OD s by subtracting the background (solvent-only/ TTC-untreated controls).

29. Use the average reading of the three technical replicates.

DATA ANALYSIS

30. Correct test data: subtract average background of the exaction solvent-only control.

31. Convert the OD to μg mL−1 formazan (Sigma Aldrich #93145) using the linear-model of standard samplese.

^*This highlights steps of the protocol which may need modified and/or standardised for seeds for other species according to their parameters such as thousand seed weight and/or the (metabolically active) embryo weight relative to that of the endosperm.

^aEnsure all of the supernatant is removed as this may interfere with OD measurements.

^bEnsure all the residue is transferred to the filter-paper.

^cEnsure that there is no loss of material during grinding in the mortar.

^dEnsure that residue is not re-suspended during pipetting.

^eThe standard samples are prepare by dissolving formazan in extraction solvent.